Where Does Electron Transfer Dissociation Occur in an Orbitrap

This article references 401 other publications.

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Journal of Proteome Research (2008), 7 (8), 3127-3136CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Here we detail the modification of a quadrupole linear ion trap-orbitrap hybrid (QLT-orbitrap) mass spectrometer to accommodate a neg. chem. ionization (NCI) source. The NCI source is used to produce fluoranthene radical anions for imparting electron transfer dissocn. (ETD). The anion beam is stable, robust, and intense so that a sufficient amt. of reagents can be injected into the QLT in only 4-8 ms. Following ion/ion reaction in the QLT, ETD product ions are mass-to-charge (m/z) analyzed in either the QLT (for speed and sensitivity) or the orbitrap (for mass resoln. and accuracy). Here we describe the phys. layout of this device, parametric optimization of anion transport, an evaluation of relevant ETD figures of merit, and the application of this instrument to protein sequence anal. Described proteomic applications include complex peptide mixt. anal., post-translational modification (PTM) site identification, isotope-encoded quantitation, large peptide characterization, and intact protein anal. From these expts., we conclude the ETD-enabled orbitrap will provide the proteomic field with several new opportunities and represents an advance in protein sequence anal. technologies.

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Molecular and Cellular Proteomics (2007), 6 (11), 1942-1951CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)

We performed a large scale study of electron transfer dissocn. (ETD) performance, as compared with ion trap collision-activated dissocn. (CAD), for peptides ranging from ∼1000 to 5000 Da (n ∼ 4000). These data indicate relatively little overlap in peptide identifications between the two methods (∼12%). ETD outperformed CAD for all charge states greater than 2; however, regardless of precursor charge a linear decrease in percent fragmentation, as a function of increasing precursor m/z, was obsd. with ETD fragmentation. We postulate that several precursor cation attributes, including peptide length, charge distribution, and total mass, could be relevant players. To examine these parameters unique ETD-identified peptides were sorted by length, and the ratio of amino acid residues per precursor charge (residues/charge) was calcd. We obsd. excellent correlation between the ratio of residues/charge and percent fragmentation. For peptides of a given residue/charge ratio, there is no correlation between peptide mass and percent fragmentation; instead we conclude that the ratio of residues/charge is the main factor in detg. a successful ETD outcome. As charge d. decreases so does the probability of non-covalent interactions that can bind a newly formed c/z-type ion pair. Recently we have described a supplemental activation approach (ETcaD) to convert these non-dissociative electron transfer product ions to useful c- and z-type ions. Automated implementation of such methods should remove this apparent precursor m/z ceiling. Finally, we evaluated the role of ion d. (both anionic and cationic) and reaction duration for an ETD expt. These data indicate that the best performance is achieved when the ion trap is filled to its space charge limit with anionic reagents. In this largest scale study of ETD to date, ETD continues to show great promise to propel the field of proteomics and, for small- to medium-sized peptides, is highly complementary to ion trap CAD.

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Nature Methods (2008), 5 (11), 959-964CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

Mass spectrometry has become a key technol. for modern large-scale protein sequencing. Tandem mass spectrometry, the process of peptide ion dissocn. followed by mass-to-charge ratio (m/z) anal., is the crit. component for peptide identification. Recent advances in mass spectrometry now permit two discrete, and complementary, types of peptide ion fragmentation: collision-activated dissocn. (CAD) and electron transfer dissocn. (ETD) on a single instrument. To exploit this complementarity and increase sequencing success rates, we designed and embedded a data-dependent decision tree algorithm (DT) to make unsupervised, real-time decisions of which fragmentation method to use based on precursor charge and m/z. Applying the DT to large-scale proteome analyses of Saccharomyces cerevisiae and human embryonic stem cells, we identified 53,055 peptides in total, which was greater than by using CAD (38,293) or ETD (39,507) alone. In addn., the DT method also identified 7422 phosphopeptides, compared to either 2801 (CAD) or 5874 (ETD) phosphopeptides.

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Frese, Christian K.; Altelaar, A. F. Maarten; Hennrich, Marco L.; Nolting, Dirk; Zeller, Martin; Griep-Raming, Jens; Heck, Albert J. R.; Mohammed, Shabaz

Journal of Proteome Research (2011), 10 (5), 2377-2388CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Over the past decade peptide sequencing by collision induced dissocn. (CID) has become the method of choice in mass spectrometry-based proteomics. The development of alternative fragmentation techniques such as electron transfer dissocn. (ETD) has extended the possibilities within tandem mass spectrometry. Recent advances in instrumentation allow peptide fragment ions to be detected with high speed and sensitivity (e.g., in a 2D or 3D ion trap) or at high resoln. and high mass accuracy (e.g., an Orbitrap or a ToF). Here, we describe a comprehensive exptl. comparison of using ETD, ion-trap CID, and beam type CID (HCD) in combination with either linear ion trap or Orbitrap readout for the large-scale anal. of tryptic peptides. We investigate which combination of fragmentation technique and mass analyzer provides the best performance for the anal. of distinct peptide populations such as N-acetylated, phosphorylated, and tryptic peptides with up to two missed cleavages. We found that HCD provides more peptide identifications than CID and ETD for doubly charged peptides. In terms of Mascot score, ETD FT outperforms the other techniques for peptides with charge states higher than 2. Our data shows that there is a trade-off between spectral quality and speed when using the Orbitrap for fragment ion detection. We conclude that a decision-tree regulated combination of higher-energy collisional dissocn. (HCD) and ETD can improve the av. Mascot score.

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Horn, David M.; Ge, Ying; McLafferty, Fred W.

Analytical Chemistry (2000), 72 (20), 4778-4784CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

In previous studies, electron capture dissocn. (ECD) has been successful only with ionized smaller proteins, cleaving between 33 of the 153 amino acid pairs of a 17 kDa protein. This has been increased to 99 cleavages by colliding the ions with a background gas while subjecting them to electron capture. Presumably this ion activation breaks intramol. noncovalent bonds of the ion's secondary and tertiary structure that otherwise prevent sepn. of the products from the nonergodic ECD cleavage of a backbone covalent bond. In comparison to collisionally activated dissocn., this "activated ion" (AI) ECD provides more extensive, and complementary, sequence information. AI ECD effected cleavage of 116, 60, and 47, resp., backbone bonds in 29, 30, and 42 kDa proteins to provide extensive contiguous sequence information on both termini; AI conditions are being sought to denature the center portion of these large ions. This accurate "sequence tag" information could potentially identify individual proteins in mixts. at far lower sample levels than methods requiring prior proteolysis.

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Horn, D. M. ; Breuker, K. ; Frank, A. J. ; McLafferty, F. W. J. Am. Chem. Soc. 2001 , 123 , 9792 – 9799 DOI: 10.1021/ja003143u

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Kinetic Intermediates in the Folding of Gaseous Protein Ions Characterized by Electron Capture Dissociation Mass Spectrometry

Horn, David M.; Breuker, Kathrin; Frank, Aaron J.; McLafferty, Fred W.

Journal of the American Chemical Society (2001), 123 (40), 9792-9799CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

Alternative mechanisms propose that protein folding in soln. proceeds either through specific obligate intermediates or by a multiplicity of routes in a folding funnel. These questions are examd. in the gas phase by using a new method that provides details of the noncovalent binding of solvent-free protein ions. Capture of an electron by a multiply charged cation causes immediate dissocn. (ECD) of a backbone bond, but with negligible excitation of noncovalent bonds; thus ECD of a linear protein ion produces two measurable fragment ions only if these are not held together by noncovalent bonds. Thermal unfolding of 9+ ions of cytochrome c proceeds through the sep. unfolding of up to 13 backbone regions (represented by 44 bond cleavages) with melting temps. of <26 to 140 °C. An 0.25 s laser IR pulse induces unfolding of 9+ ions in <4 s in six of these regions, followed by their refolding in 2 min. However, for the 15+ ions a laser IR pulse causes slower unfolding through poorly defined intermediates that leads to far more ECD products (63% increase in bond cleavages) after 1 min, even more than heating to 140 °C, with refolding to a more compact conformation in 10 min. Random isomerization appears to produce a dynamic mixt. of conformers that folds through a variety of pathways to the most stable conformer(s), consistent with a folding funnel; this might also be considered as an extension of the classical view to a system with a far smaller free energy change yielding multiple conformers. As cautions to inferring soln. conformational structure from gas-phase data, no structural relationship between these gaseous folding intermediates and those in soln. is apparent, consistent with reduced hydrophobic bonding and increased electrostatic repulsion. Further, equil. folding of gaseous ions can require minutes, and even momentary unfolding of an intermol. complex during this time can be irreversible.

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Sze, S. K. ; Ge, Y. ; Oh, H. ; McLafferty, F. W. Proc. Natl. Acad. Sci. U. S. A. 2002 , 99 , 1774 – 1779 DOI: 10.1073/pnas.251691898

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Top-down mass spectrometry of a 29-kDa protein for characterization of any posttranslational modification to within one residue

Sze, Siu Kwan; Ge, Ying; Oh, HanBin; McLafferty, Fred W.

Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (4), 1774-1779CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

A mass difference between the measured mol. wt. of a protein and that of its DNA-predicted sequence indicates sequence errors and/or posttranslational modifications. In the top-down mass spectrometry approach, the measured mol. ion is dissocd., and these fragment masses are matched against those predicted from the protein sequence to restrict the locations of the errors/modifications. The proportion of the ion's interresidue bonds that are cleaved dets. the specificity of such locations; previously, ubiquitin (76 residues) was the largest for which all such bonds were dissocd. Now, cleavages are achieved for carbonic anhydrase at 250 of the 258 interresidue locations. Cleavages of three spectra would define posttranslational modifications at 235 residues to within one residue. For 24 of the 34 possible phosphorylation sites, the cleavages of one spectrum would delineate exactly all -PO3H substitutions. This result has been achieved with electron-capture dissocn. by minimizing the further cleavage of primary product ions and by denaturing the tertiary noncovalent bonding of the mol. ions under a variety of conditions.

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Tsybin, Y. O. ; Witt, M. ; Baykut, G. ; Kjeldsen, F. ; Håkansson, P. Rapid Commun. Mass Spectrom. 2003 , 17 , 1759 – 1768 DOI: 10.1002/rcm.1118

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Combined infrared multiphoton dissociation and electron capture dissociation with a hollow electron beam in Fourier transform ion cyclotron resonance mass spectrometry

Tsybin, Youri O.; Witt, Matthias; Baykut, Goekhan; Kjeldsen, Frank; Hakansson, Per

Rapid Communications in Mass Spectrometry (2003), 17 (15), 1759-1768CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)

An electron injection system based on an indirectly heated ring-shaped dispenser cathode was developed and installed in a 7 T Fourier transform ICR (FTICR) mass spectrometer. This new hardware design allows high-rate electron capture dissocn. (ECD) to be carried out by a hollow electron beam coaxial with the ICR trap. IR multiphoton dissocn. (IRMPD) can also be performed with an on-axis IR-laser beam passing through a hole at the center of the dispenser cathode. Electron and photon irradn. times of the order of 100 ms are required for efficient ECD and IRMPD, resp. As ECD and IRMPD generate fragments of different types (mostly c, z and b, y, resp.), complementary structural information that improves the characterization of peptides and proteins by FTICR mass spectrometry can be obtained. The developed technique enables the consecutive or simultaneous use of the ECD and IRMPD methods within a single FTICR exptl. sequence and on the same ensemble of trapped ions in multistage tandem (MS/MS/MS or MSn) mass spectrometry. Flexible changing between ECD and IRMPD should present advantages for the anal. of protein digests sepd. by liq. chromatog. prior to FTICRMS. Also, ion activation by either electron or laser irradn. prior to, as well as after, dissocn. by IRMPD or ECD increases the efficiency of ion fragmentation, including the w-type fragment ion formation, and improves sequencing of peptides with multiple disulfide bridges. The developed instrumental configuration is essential for combined ECD and IRMPD on FTICR mass spectrometers with limited access into the ICR trap.

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Cooper, H. J. ; Håkansson, K. ; Marshall, A. G. Mass Spectrom. Rev. 2005 , 24 , 201 – 222 DOI: 10.1002/mas.20014

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The role of electron capture dissociation in biomolecular analysis

Cooper, Helen J.; Hakansson, Kristina; Marshall, Alan G.

Mass Spectrometry Reviews (2005), 24 (2), 201-222CODEN: MSRVD3; ISSN:0277-7037. (John Wiley & Sons, Inc.)

A review. The introduction of electron capture dissocn. (ECD) to electrospray (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) constitutes a significant advance in the structural anal. of biomols. The fundamental features and benefits of ECD are discussed in this review. ECD is currently unique to FT-ICR MS and the fundamentals of that technique are outlined. The advantages and complementarity of ECD in relation to other tandem mass spectrometry (MS/MS) techniques, such as IR multiphoton dissocn. (IRMPD) and sustained off-resonance collision-induced dissocn. (SORI-CID), are discussed. The instrumental considerations assocd. with implementation of ECD, including activated ion techniques and coupling to online sepn. techniques, are covered, as are the allied processes electronic excitation dissocn. (EED), electron detachment dissocn. (EDD), and hot electron capture (HECD). A major theme of this review is the role of ECD in proteomics, particularly for characterization of post-translational modifications (phosphorylation, glycosylation, carboxyglutamic acid, sulfation, acylation, and methionine oxidn.) and the top-down approach to protein identification. The application of ECD to the anal. of polymers, peptide nucleic acids, and oligonucleotides is also discussed.

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Mikhailov, V. A. ; Cooper, H. J. J. Am. Soc. Mass Spectrom. 2009 , 20 , 763 – 771 DOI: 10.1016/j.jasms.2008.12.015

McLuckey, S. A. ; Reid, G. E. ; Wells, J. M. Anal. Chem. 2002 , 74 , 336 – 346 DOI: 10.1021/ac0109671

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Ion parking during ion/ion reactions in electrodynamic ion traps

McLuckey, Scott A.; Reid, Gavin E.; Wells, J. Mitchell

Analytical Chemistry (2002), 74 (2), 336-346CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Under appropriate ion d. conditions, it is possible to selectively inhibit rates of ion/ion reactions in a quadrupole ion trap via the application of oscillatory voltages to one or more electrodes of the ion trap. The phenomenon is demonstrated using dipolar resonance excitation applied to the end-cap electrodes of a three-dimensional quadrupole ion trap. The application of a resonance excitation voltage tuned to inhibit the ion/ion reaction rate of a specific range of ion mass-to-charge ratios is referred to as "ion parking". The bases for rate inhibition are (i) an increase in the relative velocity of the ion/ion reaction pair, which reduces the cross section for ion/ion capture and, at least in some cases, (ii) redn. in the time of phys. overlap of pos. charged and neg. charged ion clouds. The efficiency and specificity of the ion parking expt. is highly dependent upon ion densities, trapping conditions, ion charge states, and resonance excitation conditions. The ion parking expt. is illustrated herein along with applications to the concn. of ions originally present over a range of charge states into a selected charge state and in the selection of a particular ion from a set of ions derived from a simple protein mixt.

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Han, H. ; Xia, Y. ; McLuckey, S. A. Rapid Commun. Mass Spectrom. 2007 , 21 , 1567 – 1573 DOI: 10.1002/rcm.2994

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Beam-type collisional activation of polypeptide cations that survive ion/ion electron transfer

Han, Hongling; Xia, Yu; McLuckey, Scott A.

Rapid Communications in Mass Spectrometry (2007), 21 (10), 1567-1573CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)

Doubly protonated peptides that undergo an electron transfer reaction without dissocn. in a linear ion trap can be subjected to beam-type collisional activation upon transfer from the linear ion trap into an adjacent mass analyzer, as demonstrated here with a hybrid triple quadrupole/linear ion trap system. The activation can be promoted by use of a DC offset difference between the ion trap used for reaction and the ion trap into which the products are injected of 12-16 V, which gives rise to energetic collisions between the transferred ions and the collision/bath gas employed in the linear ion trap used for ion/ion reactions. Such a process can be executed routinely on hybrid linear ion trap/triple quadrupole tandem mass spectrometers and is demonstrated here with several model peptides as well as a few dozen tryptic peptides. Collisional activation of the peptide precursor ions that survive electron transfer frequently provides structural information that is absent from the precursor ions that fragment spontaneously upon electron transfer. The degree to which addnl. structural information is obtained by collisional activation of the surviving singly charged peptide ions depends upon peptide size. Little or no addnl. structural information is obtained from small peptides (<8 residues) due to the high electron transfer dissocn. (ETD) efficiencies noted for these peptides as well as the extensive sequence information that tends to be forthcoming from ETD of such species. Collisional activation of the surviving electron transfer products provided greatest benefit for peptides of 8-15 residues.

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Wu, S.-L. ; Hühmer, A. F. R. ; Hao, Z. ; Karger, B. L. J. Proteome Res. 2007 , 6 , 4230 – 4244 DOI: 10.1021/pr070313u

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On-Line LC-MS Approach Combining Collision-Induced Dissociation (CID), Electron-Transfer Dissociation (ETD), and CID of an Isolated Charge-Reduced Species for the Trace-Level Characterization of Proteins with Post-Translational Modifications

Wu, Shiaw-Lin; Huehmer, Andreas F. R.; Hao, Zhiqi; Karger, Barry L.

Journal of Proteome Research (2007), 6 (11), 4230-4244CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

The authors have expanded their recent online LC-MS platform for large peptide anal. to combine collision-induced dissocn. (CID), electron-transfer dissocn. (ETD), and CID of an isolated charge-reduced (CRCID) species derived from ETD to det. sites of phosphorylation and glycosylation modifications, as well as the sequence of large peptide fragments (i.e., 2000-10,000 Da) from complex proteins, such as β-casein, epidermal growth factor receptor (EGFR), and tissue plasminogen activator (t-PA) at the low femtomol level. The incorporation of an addnl. CID activation step for a charge-reduced species, isolated from ETD fragment ions, improved ETD fragmentation when precursor ions with high m/z (approx. >1000) were automatically selected for fragmentation. Specifically, the identification of the exact phosphorylation sites was strengthened by the extensive coverage of the peptide sequence with a near-continuous product ion series. The identification of N-linked glycosylation sites in EGFR and an O-linked glycosylation site in t-PA were also improved through the enhanced identification of the peptide backbone sequence of the glycosylated precursors. The new strategy is a good starting survey scan to characterize enzymic peptide mixts. over a broad range of masses using LC-MS with data-dependent acquisition, as the three activation steps can provide complementary information to each other. In general, large peptides can be extensively characterized by the ETD and CRCID steps, including sites of modification from the generated, near-continuous product ion series, supplemented by the CID-MS2 step. At the same time, small peptides (e.g., ≤2+ ions), which lack extensive ETD or CRCID fragmentation, can be characterized by the CID-MS2 step. A more targeted approach can then be followed in subsequent LC-MS runs to obtain addnl. information, if needed. Overall, the recently introduced ETD not only provides useful structural information, but also enhances the confidence of all assignments. The sensitivity of this new approach on the chromatog. time scale is similar to the previous Extended Range Proteomic Anal. (ERPA) using CID-MS2 and CID-MS3. The new LC-MS platform can be anticipated to be a useful approach for the comprehensive characterization of complex proteins.

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Xia, Y. ; Han, H. ; McLuckey, S. A. Anal. Chem. 2008 , 80 , 1111 – 1117 DOI: 10.1021/ac702188q

Liu, J. ; Gunawardena, H. P. ; Huang, T. Y. ; McLuckey, S. A. Int. J. Mass Spectrom. 2008 , 276 , 160 – 170 DOI: 10.1016/j.ijms.2008.07.028

Campbell, J. L. ; Hager, J. W. ; Le Blanc, J. C. Y. J. Am. Soc. Mass Spectrom. 2009 , 20 , 1672 – 1683 DOI: 10.1016/j.jasms.2009.05.009

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On Performing Simultaneous Electron Transfer Dissociation and Collision-Induced Dissociation on Multiply Protonated Peptides in a Linear Ion Trap

Campbell, J. Larry; Hager, James W.; Le Blanc, J. C. Yves

Journal of the American Society for Mass Spectrometry (2009), 20 (9), 1672-1683CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)

The authors propose a tandem mass spectrometry method that combines electron-transfer dissocn. (ETD) with simultaneous collision-induced dissocn. (CID), termed ETD/CID. This technique can provide more complete sequence coverage of peptide ions, esp. those at lower charge states. A selected precursor ion is isolated and subjected to ETD. At the same time, a residual precursor ion is subjected to activation via CID. The specific residual precursor ion selected for activation will depend upon the charge state and m/z of the ETD precursor ion. Residual precursor ions, which include unreacted precursor ions and charge-reduced precursor ions (either by electron-transfer or proton transfer), are often abundant remainders in ETD-only reactions. Preliminary results demonstrate that during an ETD/CID expt., b, y, c, and z-type ions can be produced in a single expt. and displayed in a single mass spectrum. While some peptides, esp. doubly protonated ones, do not fragment well by ETD, ETD/CID alleviates this problem by acting in at least one of three ways: (1) the no. of ETD fragment ions are enhanced by CID of residual precursor ions, (2) both ETD and CID-derived fragments are produced, or (3) predominantly CID-derived fragments are produced with little or no improvement in ETD-derived fragment ions. Two interesting scenarios are presented that display the flexibility of the ETD/CID method. For example, smaller peptides that show little response to ETD are fragmented preferentially by CID during the ETD/CID expt. Conversely, larger peptides with higher charge states are fragmented primarily via ETD. Hence, ETD/CID appears to rely upon the fundamental reactivity of the analyte cations to provide the best fragmentation without implementing any addnl. logic or MS/MS expts. In addn. to the ETD/CID expts., the authors describe a novel dual source interface for providing front-end ETD capabilities on a linear ion trap mass spectrometer.

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Rathore, D. ; Aboufazeli, F. ; Dodds, E. D. Analyst 2015 , 140 , 7175 – 7183 DOI: 10.1039/C5AN01225B

Swaney, D. L. ; McAlister, G. C. ; Wirtala, M. ; Schwartz, J. C. ; Syka, J. E. P. ; Coon, J. J. Anal. Chem. 2007 , 79 , 477 – 485 DOI: 10.1021/ac061457f

Frese, C. K. ; Altelaar, A. F. M. ; van den Toorn, H. ; Nolting, D. ; Griep-Raming, J. ; Heck, A. J. R. ; Mohammed, S. Anal. Chem. 2012 , 84 , 9668 – 9673 DOI: 10.1021/ac3025366

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Toward Full Peptide Sequence Coverage by Dual Fragmentation Combining Electron-Transfer and Higher-Energy Collision Dissociation Tandem Mass Spectrometry

Frese, Christian K.; Altelaar, A. F. Maarten; van den Toorn, Henk; Nolting, Dirk; Griep-Raming, Jens; Heck, Albert J. R.; Mohammed, Shabaz

Analytical Chemistry (Washington, DC, United States) (2012), 84 (22), 9668-9673CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Increasing peptide sequence coverage by tandem mass spectrometry improves confidence in database search-based peptide identification and facilitates mapping of post-translational modifications and de novo sequencing. Inducing 2-fold fragmentation by combining electron-transfer and higher-energy collision dissocn. (EThcD) generates dual fragment ion series and facilitates extensive peptide backbone fragmentation. After an initial electron-transfer dissocn. step, all ions including the unreacted precursor ions are subjected to collision induced dissocn. which yields b/y- and c/z-type fragment ions in a single spectrum. This new fragmentation scheme provides richer spectra and substantially increases the peptide sequence coverage and confidence in peptide identification.

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O'Connor, P. B. ; Lin, C. ; Cournoyer, J. J. ; Pittman, J. L. ; Belyayev, M. ; Budnik, B. A. J. Am. Soc. Mass Spectrom. 2006 , 17 , 576 – 585 DOI: 10.1016/j.jasms.2005.12.015

Tsybin, Y. O. ; He, H. ; Emmett, M. R. ; Hendrickson, C. L. ; Marshall, A. G. Anal. Chem. 2007 , 79 , 7596 – 7602 DOI: 10.1021/ac071165u

Savitski, M. M. ; Kjeldsen, F. ; Nielsen, M. L. ; Zubarev, R. A. J. Am. Soc. Mass Spectrom. 2007 , 18 , 113 – 120 DOI: 10.1016/j.jasms.2006.09.008

Ledvina, A. R. ; McAlister, G. C. ; Gardner, M. W. ; Smith, S. I. ; Madsen, J. A. ; Schwartz, J. C. ; Stafford, G. C. ; Syka, J. E. P. ; Brodbelt, J. S. ; Coon, J. J. Angew. Chem., Int. Ed. 2009 , 48 , 8526 – 8528 DOI: 10.1002/anie.200903557

Riley, N. M. ; Westphall, M. S. ; Hebert, A. S. ; Coon, J. J. Anal. Chem. 2017 , 89 , 6358 – 6366 DOI: 10.1021/acs.analchem.7b00213

Ledvina, A. R. ; Beauchene, N. A. ; McAlister, G. C. ; Syka, J. E. P. ; Schwartz, J. C. ; Griep-Raming, J. ; Westphall, M. S. ; Coon, J. J. Anal. Chem. 2010 , 82 , 10068 – 10074 DOI: 10.1021/ac1020358

Chalkley, R. J. ; Medzihradszky, K. F. ; Lynn, A. J. ; Baker, P. R. ; Burlingame, A. L. Anal. Chem. 2010 , 82 , 579 – 584 DOI: 10.1021/ac9018582

Cannon, J. R. ; Holden, D. D. ; Brodbelt, J. S. Anal. Chem. 2014 , 86 , 10970 – 10977 DOI: 10.1021/ac5036082

Michalski, A. ; Damoc, E. ; Lange, O. ; Denisov, E. ; Nolting, D. ; Müller, M. ; Viner, R. ; Schwartz, J. ; Remes, P. ; Belford, M. ; Dunyach, J.-J. ; Cox, J. ; Horning, S. ; Mann, M. ; Makarov, A. Mol. Cell. Proteomics 2012 , 11 , O111.013698 DOI: 10.1074/mcp.O111.013698

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Ultra high resolution linear ion trap orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes

Michalski, Annette; Damoc, Eugen; Lange, Oliver; Denisov, Eduard; Nolting, Dirk; Mueller, Mathias; Viner, Rosa; Schwartz, Jae; Remes, Philip; Belford, Michael; Dunyach, Jean-Jacques; Cox, Juergen; Horning, Stevan; Mann, Matthias; Makarov, Alexander

Molecular and Cellular Proteomics (2012), 11 (3), 013698/1-013698/11CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the std. mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the obsd. frequencies. An enhanced Fourier Transform algorithm-incorporating phase information-further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down expts., we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liq. chromatog. tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissocn. of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixts. by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissocn. mechanisms are routinely obtained of all fragmented peptides.

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Rose, C. M. ; Russell, J. D. ; Ledvina, A. R. ; McAlister, G. C. ; Westphall, M. S. ; Griep-Raming, J. ; Schwartz, J. C. ; Coon, J. J. ; Syka, J. E. P. J. Am. Soc. Mass Spectrom. 2013 , 24 , 816 – 827 DOI: 10.1007/s13361-013-0622-0

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Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

Rose, Christopher M.; Russell, Jason D.; Ledvina, Aaron R.; McAlister, Graeme C.; Westphall, Michael S.; Griep-Raming, Jens; Schwartz, Jae C.; Coon, Joshua J.; Syka, John E. P.

Journal of the American Society for Mass Spectrometry (2013), 24 (6), 816-827CODEN: JAMSEF; ISSN:1044-0305. (Springer)

The authors describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. The authors partitioned the collision cell into a multi-section radiofrequency ion storage and transfer device to enable injection and simultaneous sep. storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell and lens electrodes enables charge-sign independent trapping for ion-ion reactions. The ∼2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell's longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z anal. of the ETD product ions. This extends the intra-scan dynamic range by increasing the max. no. of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, the authors demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS anal. of intact protein species as compared to the std. ETD implementation.

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Ledvina, A. R. ; Rose, C. M. ; McAlister, G. C. ; Syka, J. E. P. ; Westphall, M. S. ; Griep-Raming, J. ; Schwartz, J. C. ; Coon, J. J. J. Am. Soc. Mass Spectrom. 2013 , 24 , 1623 – 1633 DOI: 10.1007/s13361-013-0621-1

Frese, C. K. ; Nolting, D. ; Altelaar, A. F. M. ; Griep-Raming, J. ; Mohammed, S. ; Heck, A. J. R. J. Am. Soc. Mass Spectrom. 2013 , 24 , 1663 – 1670 DOI: 10.1007/s13361-013-0618-9

Earley, L. ; Anderson, L. C. ; Bai, D. L. ; Mullen, C. ; Syka, J. E. P. ; English, A. M. ; Dunyach, J.-J. ; Stafford, G. C. ; Shabanowitz, J. ; Hunt, D. F. ; Compton, P. D. ; Compton, P. D. Anal. Chem. 2013 , 85 , 8385 – 8390 DOI: 10.1021/ac401783f

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Front-End Electron Transfer Dissociation: A New Ionization Source

Earley, Lee; Anderson, Lissa C.; Bai, Dina L.; Mullen, Christopher; Syka, John E. P.; English, A. Michelle; Dunyach, Jean-Jacques; Stafford, George C.; Shabanowitz, Jeffrey; Hunt, Donald F.; Compton, Philip D.

Analytical Chemistry (Washington, DC, United States) (2013), 85 (17), 8385-8390CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Electron transfer dissocn. (ETD), a technique that provides efficient fragmentation while depositing little energy into vibrational modes, has been widely integrated into proteomics workflows. Current implementations of this technique, as well as other ion-ion reactions like proton transfer, involve sophisticated hardware, lack robustness, and place severe design limitations on the instruments to which they are attached. Described herein is a novel, elec. discharge-based reagent ion source that is located in the first differentially pumped region of the mass spectrometer. The reagent source was found to produce intense reagent ion signals over extended periods of time while having no measurable impact on precursor ion signal. Further, the source is simple to construct and enables implementation of ETD on any instrument without modification to footprint. Finally, in the context of hybrid mass spectrometers, relocation of the reagent ion source to the front of the mass spectrometer enables new approaches to gas phase interrogation of intact proteins.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1Wis7%252FM&md5=e4fc5c58a545d35320bb063148d163e1

Senko, M. W. ; Remes, P. M. ; Canterbury, J. D. ; Mathur, R. ; Song, Q. ; Eliuk, S. M. ; Mullen, C. ; Earley, L. ; Hardman, M. ; Blethrow, J. D. ; Bui, H. ; Specht, A. ; Lange, O. ; Denisov, E. ; Makarov, A. ; Horning, S. ; Zabrouskov, V. Anal. Chem. 2013 , 85 , 11710 – 11714 DOI: 10.1021/ac403115c

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Novel Parallelized Quadrupole/Linear Ion Trap/Orbitrap Tribrid Mass Spectrometer Improving Proteome Coverage and Peptide Identification Rates

Senko, Michael W.; Remes, Philip M.; Canterbury, Jesse D.; Mathur, Raman; Song, Qingyu; Eliuk, Shannon M.; Mullen, Chris; Earley, Lee; Hardman, Mark; Blethrow, Justin D.; Bui, Huy; Specht, August; Lange, Oliver; Denisov, Eduard; Makarov, Alexander; Horning, Stevan; Zabrouskov, Vlad

Analytical Chemistry (Washington, DC, United States) (2013), 85 (24), 11710-11714CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Proteome coverage and peptide identification rates have historically advanced in line with improvements to the detection limits and acquisition rate of the mass spectrometer. For a linear ion trap/Orbitrap hybrid, the acquisition rate has been limited primarily by the duration of the ion accumulation and anal. steps. The spectral acquisition rate can be significantly improved through extensive parallelization of the acquisition process using a novel mass spectrometer incorporating quadrupole, Orbitrap, and linear trap analyzers. Further, these improvements to the acquisition rate continue to enhance proteome coverage and general exptl. throughput.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsl2nt7rN&md5=104eb579a41faf12a0d41a2a8148e18f

Riley, N. M. ; Mullen, C. ; Weisbrod, C. R. ; Sharma, S. ; Senko, M. W. ; Zabrouskov, V. ; Westphall, M. S. ; Syka, J. E. P. ; Coon, J. J. J. Am. Soc. Mass Spectrom. 2016 , 27 , 520 – 531 DOI: 10.1007/s13361-015-1306-8

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Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

Riley, Nicholas M.; Mullen, Christopher; Weisbrod, Chad R.; Sharma, Seema; Senko, Michael W.; Zabrouskov, Vlad; Westphall, Michael S.; Syka, John E. P.; Coon, Joshua J.

Journal of the American Society for Mass Spectrometry (2016), 27 (3), 520-531CODEN: JAMSEF; ISSN:1044-0305. (Springer)

Electron transfer dissocn. (ETD) is a valuable tool for protein sequence anal., esp. for the fragmentation of intact proteins. However, low product ion signal-to-noise often requires some degree of signal averaging to achieve high quality MS/MS spectra of intact proteins. Here the authors describe a new implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid, the Orbitrap Fusion Lumos, for improved product ion signal-to-noise via ETD reactions on larger precursor populations. In this new high precursor capacity ETD implementation, precursor cations are accumulated in the center section of the high pressure cell in the dual pressure linear ion trap prior to charge-sign independent trapping, rather than precursor ion sequestration in only the back section as is done for std. ETD. This new scheme increases the charge capacity of the precursor accumulation event, enabling storage of ∼3-fold more precursor charges. High capacity ETD boosts the no. of matching fragments identified in a single MS/MS event, reducing the need for spectral averaging. These improvements in intra-scan dynamic range via reaction of larger precursor populations, which have been previously demonstrated through custom modified hardware, are now available on a com. platform, offering considerable benefits for intact protein anal. and top down proteomics. The authors characterize the advantages of high precursor capacity ETD through studies with myoglobin and carbonic anhydrase.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvVygtLjM&md5=557d2afb472894bdfe4a0119a0bff501

Rose, C. M. ; Rush, M. J. P. ; Riley, N. M. ; Merrill, A. E. ; Kwiecien, N. W. ; Holden, D. D. ; Mullen, C. ; Westphall, M. S. ; Coon, J. J. J. Am. Soc. Mass Spectrom. 2015 , 26 , 1848 – 1857 DOI: 10.1007/s13361-015-1183-1

Kaplan, D. A. ; Hartmer, R. ; Speir, J. P. ; Stoermer, C. ; Gumerov, D. ; Easterling, M. L. ; Brekenfeld, A. ; Kim, T. ; Laukien, F. ; Park, M. A. Rapid Commun. Mass Spectrom. 2008 , 22 , 271 – 278 DOI: 10.1002/rcm.3356

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Electron transfer dissociation in the hexapole collision cell of a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer

Kaplan, Desmond A.; Hartmer, Ralf; Speir, J. Paul; Stoermer, Carsten; Gumerov, Dmitry; Easterling, Michael L.; Brekenfeld, Andreas; Kim, Taeman; Laukien, Frank; Park, Melvin A.

Rapid Communications in Mass Spectrometry (2008), 22 (3), 271-278CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)

Electron transfer dissocn. (ETD) of proteins is demonstrated in a hybrid quadrupole-hexapole Fourier transform ICR mass spectrometer (Qh-FTICRMS). Analyte ions are selected in the mass analyzing quadrupole, accumulated in the hexapole linear ion trap, reacted with fluoranthene reagent anions, and then analyzed via an FTICR mass analyzer. The hexapole trap allows for a broad fragment ion mass range and a high ion storage capacity. Using a 3 T FTICRMS, resolns. of 60,000 were achieved with mass accuracies averaging <1.4 ppm. The high resoln., high mass accuracy ETD spectra provided by FTICR obviates the need for proton transfer reaction (PTR) charge state redn. of ETD product ions when analyzing proteins or large peptides. This is demonstrated with the ETD of ubiquitin and apomyoglobin yielding sequence coverages of 37 and 20%, resp. The authors believe this represents the first reported successful combination of ETD and a FTICRMS.

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Xia, Y. ; Chrisman, P. A. ; Erickson, D. E. ; Liu, J. ; Liang, X. ; Londry, F. A. ; Yang, M. J. ; McLuckey, S. A. Anal. Chem. 2006 , 78 , 4146 – 4154 DOI: 10.1021/ac0606296

Hendrickson, C. L. ; Quinn, J. P. ; Kaiser, N. K. ; Smith, D. F. ; Blakney, G. T. ; Chen, T. ; Marshall, A. G. ; Weisbrod, C. R. ; Beu, S. C. J. Am. Soc. Mass Spectrom. 2015 , 26 , 1626 – 1632 DOI: 10.1007/s13361-015-1182-2

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21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: A National Resource for Ultrahigh Resolution Mass Analysis

Hendrickson, Christopher L.; Quinn, John P.; Kaiser, Nathan K.; Smith, Donald F.; Blakney, Greg T.; Chen, Tong; Marshall, Alan G.; Weisbrod, Chad R.; Beu, Steven C.

Journal of the American Society for Mass Spectrometry (2015), 26 (9), 1626-1632CODEN: JAMSEF; ISSN:1044-0305. (Springer)

We describe the design and initial performance of the first 21 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The 21 T magnet is the highest field superconducting magnet ever used for FT-ICR and features high spatial homogeneity, high temporal stability, and negligible liq. helium consumption. The instrument includes a com. dual linear quadrupole trap front end that features high sensitivity, precise control of trapped ion no., and collisional and electron transfer dissocn. A third linear quadrupole trap offers high ion capacity and ejection efficiency, and rf quadrupole ion injection optics deliver ions to a novel dynamically harmonized ICR cell. Mass resolving power of 150,000 (m/Δm50%) is achieved for bovine serum albumin (66 kDa) for a 0.38 s detection period, and greater than 2,000,000 resolving power is achieved for a 12 s detection period. Externally calibrated broadband mass measurement accuracy is typically less than 150 ppb rms, with resolving power greater than 300,000 at m/z 400 for a 0.76 s detection period. Combined anal. of electron transfer and collisional dissocn. spectra results in 68% sequence coverage for carbonic anhydrase. The instrument is part of the NSF High-Field FT-ICR User Facility and is available free of charge to qualified users.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVKmsrnE&md5=6a11f1e56cbcae425bddeb691d8b6b0a

Weisbrod, C. R. ; Kaiser, N. K. ; Syka, J. E. P. ; Early, L. ; Mullen, C. ; Dunyach, J. J. ; English, A. M. ; Anderson, L. C. ; Blakney, G. T. ; Shabanowitz, J. ; Hendrickson, C. L. ; Marshall, A. G. ; Hunt, D. F. J. Am. Soc. Mass Spectrom. 2017 , 28 , 1787 – 1795 DOI: 10.1007/s13361-017-1702-3

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Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis

Weisbrod, Chad R.; Kaiser, Nathan K.; Syka, John E. P.; Early, Lee; Mullen, Christopher; Dunyach, Jean-Jacques; English, A. Michelle; Anderson, Lissa C.; Blakney, Greg T.; Shabanowitz, Jeffrey; Hendrickson, Christopher L.; Marshall, Alan G.; Hunt, Donald F.

Journal of the American Society for Mass Spectrometry (2017), 28 (9), 1787-1795CODEN: JAMSEF; ISSN:1044-0305. (Springer)

High resoln. mass spectrometry is a key technol. for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissocn. (FETD) with a custom-built 21 T FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., ∼60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device sep. from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass anal., which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein mol. wt. and min. no. of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the no. of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down anal. are discussed.

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Zheng, X. ; Wojcik, R. ; Zhang, X. ; Ibrahim, Y. M. ; Burnum-Johnson, K. E. ; Orton, D. J. ; Monroe, M. E. ; Moore, R. J. ; Smith, R. D. ; Baker, E. S. Annu. Rev. Anal. Chem. 2017 , 10 , 71 – 92 DOI: 10.1146/annurev-anchem-061516-045212

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Coupling Front-End Separations, Ion Mobility Spectrometry, and Mass Spectrometry For Enhanced Multidimensional Biological and Environmental Analyses

Zheng Xueyun; Wojcik Roza; Ibrahim Yehia M; Burnum-Johnson Kristin E; Orton Daniel J; Monroe Matthew E; Moore Ronald J; Smith Richard D; Baker Erin S; Zhang Xing

Annual review of analytical chemistry (Palo Alto, Calif.) (2017), 10 (1), 71-92 ISSN:.

Ion mobility spectrometry (IMS) is a widely used analytical technique for rapid molecular separations in the gas phase. Though IMS alone is useful, its coupling with mass spectrometry (MS) and front-end separations is extremely beneficial for increasing measurement sensitivity, peak capacity of complex mixtures, and the scope of molecular information available from biological and environmental sample analyses. In fact, multiple disease screening and environmental evaluations have illustrated that the IMS-based multidimensional separations extract information that cannot be acquired with each technique individually. This review highlights three-dimensional separations using IMS-MS in conjunction with a range of front-end techniques, such as gas chromatography, supercritical fluid chromatography, liquid chromatography, solid-phase extractions, capillary electrophoresis, field asymmetric ion mobility spectrometry, and microfluidic devices. The origination, current state, various applications, and future capabilities of these multidimensional approaches are described in detail to provide insight into their uses and benefits.

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Williams, J. P. ; Brown, J. M. ; Campuzano, I. ; Sadler, P. J. Chem. Commun. 2010 , 46 , 5458 – 5460 DOI: 10.1039/c0cc00358a

Lermyte, F. ; Verschueren, T. ; Brown, J. M. ; Williams, J. P. ; Valkenborg, D. ; Sobott, F. Methods 2015 , 89 , 22 – 29 DOI: 10.1016/j.ymeth.2015.05.019

Cramer, C. N. ; Brown, J. M. ; Tomczyk, N. ; Nielsen, P. K. ; Haselmann, K. F. J. Am. Soc. Mass Spectrom. 2017 , 28 , 384 – 388 DOI: 10.1007/s13361-016-1538-2

Baird, M. A. ; Shvartsburg, A. A. J. Am. Soc. Mass Spectrom. 2016 , 27 , 2064 – 2070 DOI: 10.1007/s13361-016-1498-6

Donohoe, G. C. ; Maleki, H. ; Arndt, J. R. ; Khakinejad, M. ; Yi, J. ; McBride, C. ; Nurkiewicz, T. R. ; Valentine, S. J. Anal. Chem. 2014 , 86 , 8121 – 8128 DOI: 10.1021/ac501527y

Chen, B. ; Lietz, C. B. ; Li, L. Anal. Bioanal. Chem. 2017 , 1 – 11 DOI: 10.1007/s00216-017-0611-4

Marshall, D. D. ; Inutan, E. D. ; Wang, B. ; Liu, C.-W. ; Thawoos, S. ; Wager-Miller, J. ; Mackie, K. ; Trimpin, S. Proteomics 2016 , 16 , 1695 – 1706 DOI: 10.1002/pmic.201500530

He, M. ; Jiang, Y. ; Guo, D. ; Xiong, X. ; Fang, X. ; Xu, W. J. Am. Soc. Mass Spectrom. 2017 , 28 , 1262 – 1270 DOI: 10.1007/s13361-016-1504-z

Martens, J. ; Berden, G. ; Gebhardt, C. R. ; Oomens, J. Rev. Sci. Instrum. 2016 , 87 , 103108 DOI: 10.1063/1.4964703

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Infrared ion spectroscopy in a modified quadrupole ion trap mass spectrometer at the FELIX free electron laser laboratory

Martens, Jonathan; Berden, Giel; Gebhardt, Christoph R.; Oomens, Jos

Review of Scientific Instruments (2016), 87 (10), 103108/1-103108/8CODEN: RSINAK; ISSN:0034-6748. (American Institute of Physics)

Modifications made to a Paul-type quadrupole ion trap mass spectrometer is reported and its application in IR ion spectroscopy expts. is discussed. Main modifications involve optical access to the trapped ions and hardware and software coupling to a variety of IR laser sources at the FELIX IR free electron laser lab. In comparison to previously described IR ion spectroscopy expts. at the FELIX lab., significant improvements in efficiency and sensitivity were found. Effects of the trapping conditions of the ions on the IR multiple photon dissocn. spectra are explored. Enhanced photo-dissocn. is found at lower pressures in the ion trap. Spectra obtained under reduced pressure conditions more closely mimic those obtained in the high-vacuum conditions of an Fourier transform ICR mass spectrometer. A gas-mixing system is described enabling the controlled addn. of a secondary gas into He buffer gas flowing into the trap and allows for ion/mol. reactions in the trap. The electron transfer dissocn. (ETD) option of the mass spectrometer allows for IR structure characterization of ETD-generated peptide dissocn. products. (c) 2016 American Institute of Physics.

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Martens, J. ; Grzetic, J. ; Berden, G. ; Oomens, J. Nat. Commun. 2016 , 7 , 11754 DOI: 10.1038/ncomms11754

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Structural identification of electron transfer dissociation products in mass spectrometry using infrared ion spectroscopy

Martens, Jonathan; Grzetic, Josipa; Berden, Giel; Oomens, Jos

Nature Communications (2016), 7 (), 11754CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)

Tandem mass spectrometry occupies a principle place among modern anal. methods and drives many developments in the primeomicsprime sciences. Electron attachment induced dissocn. methods, as alternatives for collision-induced dissocn. have profoundly influenced the field of proteomics, enabling among others the top-down sequencing of entire proteins and the anal. of post-translational modifications. The technique, however, produces more complex mass spectra and its radical-driven reaction mechanisms remain incompletely understood. Here we demonstrate the facile structural characterization of electron transfer dissocn. generated peptide fragments by IR ion spectroscopy using the tunable free-electron laser FELIX, aiding the elucidation of the underlying dissocn. mechanisms. We apply this method to verify and revise previously proposed product ion structures for an often studied model tryptic peptide, [AlaAlaHisAlaArg+2H]2+. Comparing expt. with theory reveals that structures that would be assigned using only theor. thermodn. considerations often do not correspond to the exptl. sampled species.

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Martens, J. ; Berden, G. ; Oomens, J. Anal. Chem. 2016 , 88 , 6126 – 6129 DOI: 10.1021/acs.analchem.6b01483

Munshi, M. U. ; Craig, S. M. ; Berden, G. ; Martens, J. ; DeBlase, A. F. ; Foreman, D. J. ; McLuckey, S. A. ; Oomens, J. ; Johnson, M. A. J. Phys. Chem. Lett. 2017 , 8 , 5047 – 5052 DOI: 10.1021/acs.jpclett.7b02223

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Preparation of Labile Ni+(cyclam) Cations in the Gas Phase Using Electron-Transfer Reduction through Ion-Ion Recombination in an Ion Trap and Structural Characterization with Vibrational Spectroscopy

Munshi, Musleh U.; Craig, Stephanie M.; Berden, Giel; Martens, Jonathan; De Blase, Andrew F.; Foreman, David J.; McLuckey, Scott A.; Oomens, Jos; Johnson, Mark A.

Journal of Physical Chemistry Letters (2017), 8 (20), 5047-5052CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)

Gas-phase ion chem. methods that capture and characterize the degree of activation of small mols. in the active sites of homogeneous catalysts form a powerful new tool to unravel how ligand environments affect reactivity. A key roadblock in this development, however, is the ability to generate the fragile metal oxidn. states that are essential for catalytic activity. Here we demonstrate the prepn. of the key Ni(I) center in the widely used cyclam scaffold using ion-ion recombination as a gas-phase alternative to electrochem. redn. The singly charged Ni+(cyclam) coordination complex is generated by electron transfer from fluoranthene and azobenzene anions to doubly charged Ni2+(cyclam), using the electron-transfer dissocn. protocol in a com. quadrupole ion trap instrument and in a custom-built octopole RF ion trap. The successful prepn. of the Ni+(cyclam) cation is verified through anal. of its vibrational spectrum obtained using the IR free electron laser FELIX.

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Smith, L. M. ; Kelleher, N. L. Nat. Methods 2013 , 10 , 186 – 187 DOI: 10.1038/nmeth.2369

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Proteoform: a single term describing protein complexity

Smith, Lloyd M.; Kelleher, Neil L.; Linial, Michal; Goodlett, David; Langridge-Smith, Pat; Goo, Young Ah; Safford, George; Bonilla, Leo; Kruppa, George; Zubarev, Roman; Rontree, Jon; Chamot-Rooke, Julia; Garavelli, John; Heck, Albert; Loo, Joseph; Penque, Deborah; Hornshaw, Martin; Hendrickson, Christopher; Pasa-Tolic, Ljiljana; Borchers, Christoph; Chan, Dominic; Young, Nicholas; Agar, Jeffrey; Masselon, Christophe; Gross, Michael; McLafferty, Fred; Tsybin, Yury; Ge, Ying; Sanders, Ian; Langridge, James; Whitelegge, Julian; Marshall, Alan

Nature Methods (2013), 10 (3), 186-187CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

In this article the author proposes that the term proteoform be used to designate all of the different mol. forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts and post-translational modifications. The term should include all post-translational modifications in the PSI-MOD ontol. except those classified as reagent-derivatized or isotope-labeled residues.

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Toby, T. K. ; Fornelli, L. ; Kelleher, N. L. Annu. Rev. Anal. Chem. 2016 , 9 , 499 – 519 DOI: 10.1146/annurev-anchem-071015-041550

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Progress in Top-Down Proteomics and the Analysis of Proteoforms

Toby, Timothy K.; Fornelli, Luca; Kelleher, Neil L.

Annual Review of Analytical Chemistry (2016), 9 (), 499-519CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews)

From a mol. perspective, enactors of function in biol. are intact proteins that can be variably modified at the genetic, transcriptional, or post-translational level. Over the past 30 years, mass spectrometry (MS) has become a powerful method for the anal. of proteomes. Prevailing bottom-up proteomics operates at the level of the peptide, leading to issues with protein inference, connectivity, and incomplete sequence/modification information. Top-down proteomics (TDP), alternatively, applies MS at the proteoform level to analyze intact proteins with diverse sources of intramol. complexity preserved during anal. Fortunately, advances in prefractionation workflows, MS instrumentation, and dissocn. methods for whole-protein ions have helped TDP emerge as an accessible and potentially disruptive modality with increasingly translational value. In this review, we discuss tech. and conceptual advances in TDP, along with the growing power of proteoform-resolved measurements in clin. and translational research.

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Skinner, O. S. ; Catherman, A. D. ; Early, B. P. ; Thomas, P. M. ; Compton, P. D. ; Kelleher, N. L. Anal. Chem. 2014 , 86 , 4627 – 4634 DOI: 10.1021/ac500864w

Ahlf, D. R. ; Compton, P. D. ; Tran, J. C. ; Early, B. P. ; Thomas, P. M. ; Kelleher, N. L. J. Proteome Res. 2012 , 11 , 4308 – 4314 DOI: 10.1021/pr3004216

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Evaluation of the Compact High-Field Orbitrap for Top-Down Proteomics of Human Cells

Ahlf, Dorothy R.; Compton, Philip D.; Tran, John C.; Early, Bryan P.; Thomas, Paul M.; Kelleher, Neil L.

Journal of Proteome Research (2012), 11 (8), 4308-4314CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Mass spectrometry based proteomics generally seeks to identify and fully characterize protein species with high accuracy and throughput. Recent improvements in protein sepn. have greatly expanded the capacity of top-down proteomics (TDP) to identify a large no. of intact proteins. To date, TDP has been most tightly assocd. with Fourier transform ICR (FT-ICR) mass spectrometry. Here, the authors couple the improved sepns. to a Fourier-transform instrument based not on ICR but using the Orbitrap Elite mass analyzer. Application of this platform to H1299 human lung cancer cells resulted in the unambiguous identification of 690 unique proteins and over 2000 proteoforms identified from proteins with intact masses <50 kDa. This is an early demonstration of high throughput TDP (>500 identifications) in an Orbitrap mass spectrometer and exemplifies an accessible platform for whole protein mass spectrometry.

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Rožman, M. ; Gaskell, S. J. Rapid Commun. Mass Spectrom. 2012 , 26 , 282 – 286 DOI: 10.1002/rcm.5330

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Charge state dependent top-down characterisation using electron transfer dissociation

Rozman, Marko; Gaskell, Simon J.

Rapid Communications in Mass Spectrometry (2012), 26 (3), 282-286CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)

The dissocn. of protein ions (5-30 kDa) as a function of charge state has been explored in order to suggest the optimal charge state range for top-down sequencing. Proteins were generated under denaturing conditions and their charge states were modified via ion/ion proton transfer reactions prior to dissocn. Electron transfer dissocn. (ETD) data suggested optimal sequence coverage for charge states in the m/z range from 700 to 950 while limited sequence coverage was noted when the precursor m/z was above 1000. Sequence coverage from ETD data was found to be dependent on protein size, with smaller proteins having better sequence coverage. An obsd. depletion in sequence-related information was mainly attributed to limited instrument (ion trap) performance (m/z range and resoln.). For a combined ETD/collision-induced dissocn. (CID) approach it is difficult to propose an optimal m/z range since good sequence coverage for CID is at intermediate charge states and the optimal m/z range increases with protein size. When only one charge state can be analyzed in a combined ETD/CID approach, a range around 950 m/z is suggested as a starting point. Alternatively, two charge states should be explored, each optimal for either ETD or CID. Overall, these suggestions should be useful to achieve enhanced characterization of smaller proteins/large protein fragments (generated from denaturing solns.) in minimal anal. times. Copyright © 2011 John Wiley & Sons, Ltd.

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Tran, J. C. ; Zamdborg, L. ; Ahlf, D. R. ; Lee, J. E. ; Catherman, A. D. ; Durbin, K. R. ; Tipton, J. D. ; Vellaichamy, A. ; Kellie, J. F. ; Li, M. ; Wu, C. ; Sweet, S. M. M. ; Early, B. P. ; Siuti, N. ; LeDuc, R. D. ; Compton, P. D. ; Thomas, P. M. ; Kelleher, N. L. Nature 2011 , 480 , 254 – 258 DOI: 10.1038/nature10575

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Mapping intact protein isoforms in discovery mode using top-down proteomics

Tran, John C.; Zamdborg, Leonid; Ahlf, Dorothy R.; Lee, Ji Eun; Catherman, Adam D.; Durbin, Kenneth R.; Tipton, Jeremiah D.; Vellaichamy, Adaikkalam; Kellie, John F.; Li, Mingxi; Wu, Cong; Sweet, Steve M. M.; Early, Bryan P.; Siuti, Nertila; LeDuc, Richard D.; Compton, Philip D.; Thomas, Paul M.; Kelleher, Neil L.

Nature (London, United Kingdom) (2011), 480 (7376), 254-258CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein mols. in the body. Large-scale proteome anal. has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry. This bottom-up process affords a high no. of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, esp. when combinations of these create complex patterns of intact protein isoforms and species. Top-down interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here the authors show, using a new four-dimensional sepn. system, identification of 1043 gene products from human cells that are dispersed into more than 3000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both sepn. power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helixes. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular aging (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein mols. The technol. promises to improve the link between proteomics data and complex phenotypes in basic biol. and disease research.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlyisrnJ&md5=71693f37d374216f265d7d806f67f79f

Catherman, A. D. ; Durbin, K. R. ; Ahlf, D. R. ; Early, B. P. ; Fellers, R. T. ; Tran, J. C. ; Thomas, P. M. ; Kelleher, N. L. Mol. Cell. Proteomics 2013 , 12 , 3465 – 3473 DOI: 10.1074/mcp.M113.030114

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Large-scale Top-down Proteomics of the Human Proteome: Membrane Proteins, Mitochondria, and Senescence

Catherman, Adam D.; Durbin, Kenneth R.; Ahlf, Dorothy R.; Early, Bryan P.; Fellers, Ryan T.; Tran, John C.; Thomas, Paul M.; Kelleher, Neil L.

Molecular & Cellular Proteomics (2013), 12 (12), 3465-3473CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Top-down proteomics is emerging as a viable method for the routine identification of hundreds to thousands of proteins. In this work we report the largest top-down study to date, with the identification of 1,220 proteins from the transformed human cell line H1299 at a false discovery rate of 1̂. Multiple sepn. strategies were utilized, including the focused isolation of mitochondria, resulting in significantly improved proteome coverage relative to previous work. In all, 347 mitochondrial proteins were identified, including ∼50̂ of the mitochondrial proteome below 30 kDa and over 75̂ of the subunits constituting the large complexes of oxidative phosphorylation. Three hundred of the identified proteins were found to be integral membrane proteins contg. between 1 and 12 transmembrane helixes, requiring no specific enrichment or modified LC-MS parameters. Over 5,000 proteoforms were obsd., many harboring post-translational modifications, including over a dozen proteins contg. lipid anchors (some previously unknown) and many others with phosphorylation and methylation modifications. Comparison between untreated and senescent H1299 cells revealed several changes to the proteome, including the hyperphosphorylation of HMGA2. This work illustrates the burgeoning ability of top-down proteomics to characterize large nos. of intact proteoforms in a high-throughput fashion.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFWru73N&md5=805f4faa966c784384825fdafa04c048

Durbin, K. R. ; Fellers, R. T. ; Ntai, I. ; Kelleher, N. L. ; Compton, P. D. Anal. Chem. 2014 , 86 , 1485 – 1492 DOI: 10.1021/ac402904h

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Autopilot: An Online Data Acquisition Control System for the Enhanced High-Throughput Characterization of Intact Proteins

Durbin, Kenneth R.; Fellers, Ryan T.; Ntai, Ioanna; Kelleher, Neil L.; Compton, Philip D.

Analytical Chemistry (Washington, DC, United States) (2014), 86 (3), 1485-1492CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

The ability to study organisms by direct anal. of their proteomes without digestion via mass spectrometry has benefited greatly from recent advances in sepn. techniques, instrumentation, and bioinformatics. However, improvements to data acquisition logic have lagged in comparison. Past workflows for Top Down Proteomics (TDPs) have focused on high throughput at the expense of maximal protein coverage and characterization. This mode of data acquisition has led to enormous overlap in the identification of highly abundant proteins in subsequent LC-MS injections. Furthermore, a wealth of data is left underutilized by analyzing each newly targeted species as unique, rather than as part of a collection of fragmentation events on a distinct proteoform. Here, we present a major advance in software for acquisition of TDP data that incorporates a fully automated workflow able to detect intact masses, guide fragmentation to achieve maximal identification and characterization of intact protein species, and perform database search online to yield real-time protein identifications. On Pseudomonas aeruginosa, the software combines fragmentation events of the same precursor with previously obtained fragments to achieve improved characterization of the target form by an av. of 42 orders of magnitude in confidence. When HCD fragmentation optimization was applied to intact proteins ions, there was an 18.5 order of magnitude gain in confidence. These improved metrics set the stage for increased proteome coverage and characterization of higher order organisms in the future for sharply improved control over MS instruments in a project- and lab-wide context.

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Durbin, K. R. ; Fornelli, L. ; Fellers, R. T. ; Doubleday, P. F. ; Narita, M. ; Kelleher, N. L. J. Proteome Res. 2016 , 15 , 976 – 982 DOI: 10.1021/acs.jproteome.5b00997

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Quantitation and Identification of Thousands of Human Proteoforms below 30 kDa

Durbin, Kenneth R.; Fornelli, Luca; Fellers, Ryan T.; Doubleday, Peter F.; Narita, Masashi; Kelleher, Neil L.

Journal of Proteome Research (2016), 15 (3), 976-982CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Top-down proteomics is capable of identifying and quantitating unique proteoforms through the anal. of intact proteins. We extended the coverage of the label-free technique, achieving differential anal. of whole proteins <30 kDa from the proteomes of growing and senescent human fibroblasts. By integrating improved control software with more instrument time allocated for quantitation of intact ions, we were able to collect protein data between the two cell states, confidently comparing 1577 proteoform levels. To then identify and characterize proteoforms, our advanced acquisition software, named AUTOPILOT, employed enhanced identification efficiency in identifying 1180 unique Swiss-Prot accession nos. at 1% false-discovery rate. This coverage of the low mass proteome is equiv. to the largest previously reported but was accomplished in 23% of the total acquisition time. By maximizing both the no. of quantified proteoforms and their identification rate in an integrated software environment, this work significantly advances proteoform-resolved analyses of complex systems.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFymtro%253D&md5=b4c2a62f3212f3dbcc951345bf157fbd

Brunner, A. M. ; Lössl, P. ; Liu, F. ; Huguet, R. ; Mullen, C. ; Yamashita, M. ; Zabrouskov, V. ; Makarov, A. ; Altelaar, A. F. M. ; Heck, A. J. R. Anal. Chem. 2015 , 87 , 4152 – 4158 DOI: 10.1021/acs.analchem.5b00162

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Benchmarking Multiple Fragmentation Methods on an Orbitrap Fusion for Top-down Phospho-Proteoform Characterization

Brunner, Andrea M.; Loessl, Philip; Liu, Fan; Huguet, Romain; Mullen, Christopher; Yamashita, Masami; Zabrouskov, Vlad; Makarov, Alexander; Altelaar, A. F. Maarten; Heck, Albert J. R.

Analytical Chemistry (Washington, DC, United States) (2015), 87 (8), 4152-4158CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Top-down anal. of intact proteins by mass spectrometry provides an ideal platform for comprehensive proteoform characterization, in particular, for the identification and localization of post-translational modifications (PTM) cooccurring on a protein. One of the main bottlenecks in top-down proteomics is insufficient protein sequence coverage caused by incomplete protein fragmentation. Based on previous work on peptides, increasing sequence coverage and PTM localization by combining sequential ETD and HCD fragmentation in a single fragmentation event, the authors hypothesized that protein sequence coverage and phospho-proteoform characterization could be equally improved by this new dual fragmentation method termed EThcD, recently been made available on the Orbitrap Fusion. Here, the authors systematically benchmark the performance of several (hybrid) fragmentation methods for intact protein anal. on an Orbitrap Fusion, using as a model system a 17.5 kDa N-terminal fragment of the mitotic regulator Bora. During cell division Bora becomes multiply phosphorylated by a variety of cell cycle kinases, including Aurora A and Plk1, albeit at distinctive sites. Here, the authors monitor the phosphorylation of Bora by Aurora A and Plk1, analyzing the generated distinctive phospho-proteoforms by top-down fragmentation. EThcD and ETciD on a Fusion are feasible and capable of providing richer fragmentation spectra compared to HCD or ETD alone, increasing protein sequence coverage, and thereby facilitating phosphosite localization and the detn. of kinase specific phosphorylation sites in these phospho-proteoforms. Data are available via ProteomeXchange with identifier PXD001845.

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Riley, N. M. ; Westphall, M. S. ; Coon, J. J. Anal. Chem. 2015 , 87 , 7109 – 7116 DOI: 10.1021/acs.analchem.5b00881

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Activated Ion Electron Transfer Dissociation for Improved Fragmentation of Intact Proteins

Riley, Nicholas M.; Westphall, Michael S.; Coon, Joshua J.

Analytical Chemistry (Washington, DC, United States) (2015), 87 (14), 7109-7116CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Here we report the first implementation of activated ion electron transfer dissocn. (AI-ETD) for top down protein characterization, showing that AI-ETD definitively extends the m/z range over which ETD can be effective for fragmentation of intact proteins. AI-ETD, which leverages IR photon bombardment concurrent to the ETD reaction to mitigate nondissociative electron transfer, was performed using a novel multipurpose dissocn. cell that can perform both beam-type collisional dissocn. and ion-ion reactions on an ion trap-Orbitrap hybrid mass spectrometer. AI-ETD increased the no. of c- and z-type product ions for all charge states over ETD alone, boosting product ion yield by nearly 4-fold for low charge d. precursors. AI-ETD also outperformed HCD, generating more matching fragments for all proteins at all charge states investigated. In addn. to generating more unique fragment ions, AI-ETD provided greater protein sequence coverage compared to both HCD and ETD. In all, the effectiveness of AI-ETD across the entirety of the m/z spectrum demonstrates its efficacy for robust fragmentation of intact proteins.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVakt7vM&md5=6fbd0de4ad85a1afa1be0f513c247452

Riley, N. M. ; Westphall, M. S. ; Coon, J. J. J. Proteome Res. 2017 , 16 , 2653 – 2659 DOI: 10.1021/acs.jproteome.7b00249

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Activated Ion-Electron Transfer Dissociation Enables Comprehensive Top-Down Protein Fragmentation

Riley, Nicholas M.; Westphall, Michael S.; Coon, Joshua J.

Journal of Proteome Research (2017), 16 (7), 2653-2659CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Here the authors report the first demonstration of near-complete sequence coverage of intact proteins using activated ion-electron transfer dissocn. (AI-ETD), a method that leverages concurrent IR photoactivation to enhance electron-driven dissocn. AI-ETD produces mainly c/z-type product ions and provides comprehensive (77-97%) protein sequence coverage, outperforming HCD, ETD, and EThcD for all proteins investigated. AI-ETD also maintains this performance across precursor ion charge states, mitigating charge-state dependence that limits traditional approaches.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpslCksb8%253D&md5=0deb0d644ec3cd06aa7d432ff7377f86

Riley, N. M. ; Westphall, M. S. ; Coon, J. J. J. Am. Soc. Mass Spectrom. 2017 , DOI: 10.1007/s13361-017-1808-7

Riley, N. M. ; Hebert, A. S. ; Dürnberger, G. ; Stanek, F. ; Mechtler, K. ; Westphall, M. S. ; Coon, J. J. Anal. Chem. 2017 , 89 , 6367 – 6376 DOI: 10.1021/acs.analchem.7b00212

Shaw, J. B. ; Li, W. ; Holden, D. D. ; Zhang, Y. ; Griep-Raming, J. ; Fellers, R. T. ; Early, B. P. ; Thomas, P. M. ; Kelleher, N. L. ; Brodbelt, J. S. J. Am. Chem. Soc. 2013 , 135 , 12646 – 12651 DOI: 10.1021/ja4029654

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Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation

Shaw, Jared B.; Li, Wenzong; Holden, Dustin D.; Zhang, Yan; Griep-Raming, Jens; Fellers, Ryan T.; Early, Bryan P.; Thomas, Paul M.; Kelleher, Neil L.; Brodbelt, Jennifer S.

Journal of the American Chemical Society (2013), 135 (34), 12646-12651CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here the authors describe the implementation of 193 nm UV photodissocn. (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer's disease and in cancer pathogenesis. The 5 ns, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision- and electron-based dissocn. methods.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXotV2rsLg%253D&md5=dabb8f0fe2a56ba014b221ba3325301b

Zhao, Y. ; Riley, N. M. ; Sun, L. ; Hebert, A. S. ; Yan, X. ; Westphall, M. S. ; Rush, M. J. P. ; Zhu, G. ; Champion, M. M. ; Medie, F. M. ; Champion, P. A. D. ; Coon, J. J. ; Dovichi, N. J. Anal. Chem. 2015 , 87 , 5422 – 5429 DOI: 10.1021/acs.analchem.5b00883

Fornelli, L. ; Parra, J. ; Hartmer, R. ; Stoermer, C. ; Lubeck, M. ; Tsybin, Y. O. Anal. Bioanal. Chem. 2013 , 405 , 8505 – 8514 DOI: 10.1007/s00216-013-7267-5

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Top-down analysis of 30-80 kDa proteins by electron transfer dissociation time-of-flight mass spectrometry

Fornelli, Luca; Parra, Julien; Hartmer, Ralf; Stoermer, Carsten; Lubeck, Markus; Tsybin, Yury O.

Analytical and Bioanalytical Chemistry (2013), 405 (26), 8505-8514CODEN: ABCNBP; ISSN:1618-2642. (Springer)

Electron transfer dissocn. (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural anal. of intact proteins as large as the 80 kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resoln. and mass accuracy to simultaneously analyze intact 30-80 kDa protein ions and the complex mixt. of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of ∼30 kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, redn. of S-S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down anal. of its oxidized form.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1Cntr7O&md5=1e02ef18a05fc608531b479b2a257e6a

Anderson, L. C. ; DeHart, C. J. ; Kaiser, N. K. ; Fellers, R. T. ; Smith, D. F. ; Greer, J. B. ; LeDuc, R. D. ; Blakney, G. T. ; Thomas, P. M. ; Kelleher, N. L. ; Hendrickson, C. L. J. Proteome Res. 2017 , 16 , 1087 – 1096 DOI: 10.1021/acs.jproteome.6b00696

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Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry

Anderson, Lissa C.; DeHart, Caroline J.; Kaiser, Nathan K.; Fellers, Ryan T.; Smith, Donald F.; Greer, Joseph B.; LeDuc, Richard D.; Blakney, Greg T.; Thomas, Paul M.; Kelleher, Neil L.; Hendrickson, Christopher L.

Journal of Proteome Research (2017), 16 (2), 1087-1096CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Successful high-throughput characterization of intact proteins from complex biol. samples by mass spectrometry requires instrumentation capable of high mass resolving power, mass accuracy, sensitivity, and spectral acquisition rate. These limitations often necessitate the performance of hundreds of LC-MS/MS expts. to obtain reasonable coverage of the targeted proteome, which is still typically limited to mol. wts. <30 kDa. The National High Magnetic Field Lab. (NHMFL) recently installed a 21 T FT-ICR mass spectrometer, which is part of the NHMFL FT-ICR User Facility and available to all qualified users. Here, the authors demonstrate top-down LC-21 T FT-ICR MS/MS of intact proteins derived from human colorectal cancer cell lysate. The authors identified a combined total of 684 unique protein entries obsd. as 3238 unique proteoforms at a 1% false discovery rate, based on rapid, data-dependent acquisition of collision-induced and electron-transfer dissocn. tandem mass spectra from just 40 LC-MS/MS expts. The authors' identifications included 372 proteoforms with mol. wts. over 30 kDa detected at isotopic resoln., which substantially extends the accessible mass range for high-throughput top-down LC-MS/MS.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVGgsLnM&md5=0761405b5e1f091a684228ee791aaab7

Coelho Graça, D. ; Lescuyer, P. ; Clerici, L. ; Tsybin, Y. O. ; Hartmer, R. ; Meyer, M. ; Samii, K. ; Hochstrasser, D. F. ; Scherl, A. J. Am. Soc. Mass Spectrom. 2012 , 23 , 1750 – 1756 DOI: 10.1007/s13361-012-0446-3

Coelho Graça, D. ; Hartmer, R. ; Jabs, W. ; Beris, P. ; Clerici, L. ; Stoermer, C. ; Samii, K. ; Hochstrasser, D. ; Tsybin, Y. O. ; Scherl, A. ; Lescuyer, P. Anal. Bioanal. Chem. 2015 , 407 , 2837 – 2845 DOI: 10.1007/s00216-015-8525-5

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Identification of hemoglobin variants by top-down mass spectrometry using selected diagnostic product ions

Coelho Graca, Didia; Hartmer, Ralf; Jabs, Wolfgang; Beris, Photis; Clerici, Lorella; Stoermer, Carsten; Samii, Kaveh; Hochstrasser, Denis; Tsybin, Yury O.; Scherl, Alexander; Lescuyer, Pierre

Analytical and Bioanalytical Chemistry (2015), 407 (10), 2837-2845CODEN: ABCNBP; ISSN:1618-2642. (Springer)

Hb disorder diagnosis is a complex procedure combining several anal. steps. Due to the lack of specificity of the currently used protein anal. methods, the identification of uncommon Hb variants (proteoforms) can become a hard task to accomplish. The aim of this work was to develop a mass spectrometry-based approach to quickly identify mutated protein sequences within globin chain variants. To reach this goal, a top-down electron transfer dissocn. mass spectrometry method was developed for Hb β chain anal. A diagnostic product ion list was established with a color code strategy allowing to quickly and specifically localize a mutation in the Hb β chain sequence. The method was applied to the anal. of rare Hb β chain variants and an Aγ-β fusion protein. The results showed that the developed data anal. process allows fast and reliable interpretation of top-down electron transfer dissocn. mass spectra by nonexpert users in the clin. area.

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Sarsby, J. ; Martin, N. J. ; Lalor, P. F. ; Bunch, J. ; Cooper, H. J. J. Am. Soc. Mass Spectrom. 2014 , 25 , 1953 – 1961 DOI: 10.1007/s13361-014-0967-z

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Top-Down and Bottom-Up Identification of Proteins by Liquid Extraction Surface Analysis Mass Spectrometry of Healthy and Diseased Human Liver Tissue

Sarsby, Joscelyn; Martin, Nicholas J.; Lalor, Patricia F.; Bunch, Josephine; Cooper, Helen J.

Journal of the American Society for Mass Spectrometry (2014), 25 (11), 1953-1961CODEN: JAMSEF; ISSN:1044-0305. (Springer)

Liq. extn. surface anal. mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the anal. of thin tissue sections from human liver. The aim was to det. whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathol. Two approaches were considered. In the first, endogenous proteins were extd. from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixt. was analyzed by liq. chromatog. tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extd. by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissocn. (CID) and/or electron transfer dissocn. (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible. Top-down LESA MS anal. of healthy and diseased liver tissue revealed peaks corresponding to multiple (∼15-25) proteins. MS/MS of four of these proteins identified them as FABP1, its variant, α-Hb, and 10 kDa heat shock protein. The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathol. in sections from liver biopsies.

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Sarsby, J. ; Griffiths, R. L. ; Race, A. M. ; Bunch, J. ; Randall, E. C. ; Creese, A. J. ; Cooper, H. J. Anal. Chem. 2015 , 87 , 6794 – 6800 DOI: 10.1021/acs.analchem.5b01151

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Liquid Extraction Surface Analysis Mass Spectrometry Coupled with Field Asymmetric Waveform Ion Mobility Spectrometry for Analysis of Intact Proteins from Biological Substrates

Sarsby, Joscelyn; Griffiths, Rian L.; Race, Alan M.; Bunch, Josephine; Randall, Elizabeth C.; Creese, Andrew J.; Cooper, Helen J.

Analytical Chemistry (Washington, DC, United States) (2015), 87 (13), 6794-6800CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Previously it was shown that liq. extn. surface anal. (LESA) mass spectrometry is suitable for the anal. of intact proteins from a range of biol. substrates. LESA mass spectrometry may be coupled with high field asym. waveform ion mobility spectrometry (FAIMS) for top-down protein anal. directly from thin tissue sections (mouse liver, mouse brain) and from bacterial colonies (Escherichia coli) growing on agar. Incorporation of FAIMS results in significant improvements in signal-to-noise and reduced anal. time. Abundant protein signals are obsd. in single scan mass spectra. In addn., FAIMS enables gas-phase sepn. of mol. classes, for example, lipids and proteins, enabling improved anal. of both sets of species from a single LESA extn.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVaku7%252FL&md5=926741373ce2efaecd9bb4954442dee0

Schey, K. L. ; Anderson, D. M. ; Rose, K. L. Anal. Chem. 2013 , 85 , 6767 – 6774 DOI: 10.1021/ac400832w

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Spatially-Directed Protein Identification from Tissue Sections by Top-Down LC-MS/MS with Electron Transfer Dissociation

Schey, Kevin L.; Anderson, David M.; Rose, Kristie L.

Analytical Chemistry (Washington, DC, United States) (2013), 85 (14), 6767-6774CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for localizing both small mols. and intact proteins in a wide variety of tissue samples in both normal and diseased states. Identification of imaged signals in MALDI-IMS remains a bottleneck in the anal. and limits the interpretation of underlying biol. of tissue specimens. Spatially directed tissue microextn. of intact proteins followed by LC-MS/MS with electron transfer dissocn. (ETD) was used to identify proteins from specific locations in three tissue types; ocular lens, brain, and kidney. Detection limits were such that a 1 μL extn. vol. was sufficient to deliver proteins to the LC-MS/MS instrumentation with sufficient sensitivity to detect 50-100 proteins in a single expt. Addnl., multiple modified proteins were identified, including truncated lens proteins that would be difficult to assign to an imaged mass using a bottom-up approach. Protein sepn. and identification are expected to improve with advances in intact protein fractionation/chromatog. and advances in interpretation algorithms leading to increased depth of proteome coverage from distinct tissue locations.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXosVejsL4%253D&md5=031365b591becc1c39eba97c831c8043

100

Doll, S. ; Burlingame, A. L. ACS Chem. Biol. 2015 , 10 , 63 – 71 DOI: 10.1021/cb500904b

101

Olsen, J. V. ; Mann, M. Mol. Cell. Proteomics 2013 , 12 , 3444 – 3452 DOI: 10.1074/mcp.O113.034181

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101

Status of Large-scale Analysis of Post-translational Modifications by Mass Spectrometry

Olsen, Jesper V.; Mann, Matthias

Molecular & Cellular Proteomics (2013), 12 (12), 3444-3452CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

A review. Cellular function can be controlled through the gene expression program, but often protein post-translational modifications (PTMs) provide a more precise and elegant mechanism. Key functional roles of specific modification events-for instance, during the cell cycle-have been known for decades, but only in the past 10 years has mass-spectrometry-(MS)-based proteomics begun to reveal the true extent of the PTM universe. In this overview for the special PTM issue of Mol. and Cellular Proteomics, we take stock of where MS-based proteomics stands in the large-scale anal. of protein modifications. For many PTMs, including phosphorylation, ubiquitination, glycosylation, and acetylation, tens of thousands of sites can now be confidently identified and localized in the sequence of the protein. The quantification of PTM levels between different cellular states is likewise established, with label-free methods showing particular promise. It is also becoming possible to det. the abs. occupancy or stoichiometry of PTM sites on a large scale. Powerful software for the bioinformatic anal. of thousands of PTM sites has been developed. However, a complete inventory of sites has not been established for any PTM, and this situation will persist into the foreseeable future. Furthermore, although PTM coverage by MS-based methods is impressive, it still needs to be improved, esp. in tissues and in clin. relevant systems. The central challenge for the field is to develop streamlined methods for detg. biol. functions for the myriad of modifications now known to exist.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFWktrnI&md5=af3bdba33d24cf6b6cf31408e7162058

102

Riley, N. M. ; Coon, J. J. Anal. Chem. 2016 , 88 , 74 – 94 DOI: 10.1021/acs.analchem.5b04123

103

Humphrey, S. J. ; James, D. E. ; Mann, M. Trends Endocrinol. Metab. 2015 , 26 , 676 DOI: 10.1016/j.tem.2015.09.013

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103

Protein Phosphorylation: A Major Switch Mechanism for Metabolic Regulation

Humphrey, Sean J.; James, David E.; Mann, Matthias

Trends in Endocrinology and Metabolism (2015), 26 (12), 676-687CODEN: TENME4; ISSN:1043-2760. (Elsevier Ltd.)

A review. Metab. research is undergoing a renaissance because many diseases are increasingly recognized as being characterized by perturbations in intracellular metabolic regulation. Metabolic changes can be conferred through changes to the expression of metabolic enzymes, the concns. of substrates or products that govern reaction kinetics, or post-translational modification (PTM) of the proteins that facilitate these reactions. On the 60th anniversary since its discovery, reversible protein phosphorylation is widely appreciated as an essential PTM regulating metab. With the ability to quant. measure dynamic changes in protein phosphorylation on a global scale - hereafter referred to as phosphoproteomics - the authors are now entering a new era in metab. research, with mass spectrometry (MS)-based proteomics at the helm.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1ChtbzF&md5=df9ac75b1b38fe3eb6c1844a35e658f5

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von Stechow, L. ; Francavilla, C. ; Olsen, J. V. Expert Rev. Proteomics 2015 , 12 , 469 – 487 DOI: 10.1586/14789450.2015.1078730

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104

Recent findings and technological advances in phosphoproteomics for cells and tissues

von Stechow, Louise; Francavilla, Chiara; Olsen, Jesper V.

Expert Review of Proteomics (2015), 12 (5), 469-487CODEN: ERPXA3; ISSN:1478-9450. (Taylor & Francis Ltd.)

A review. Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins - termed phosphoproteomics - strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technol. has been applied to study a great no. of phosphorylation-based phenotypes. Nevertheless, many tech. and biol. challenges have to be overcome to identify biol. relevant phosphorylation sites in cells and tissues. This review describes different technol. strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of anal. speed and reproducibility in tissues and cells. Moreover, computational tools for anal., integration and biol. interpretation of phosphorylation events are discussed.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslCmsL3K&md5=5591e0fb5ab21e8f0f4c82622c9666aa

105

Thaysen-Andersen, M. ; Packer, N. H. ; Schulz, B. L. Mol. Cell. Proteomics 2016 , 15 , 1773 – 1790 DOI: 10.1074/mcp.O115.057638

[Crossref], [PubMed], [CAS], Google Scholar

105

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

Thaysen-Andersen, Morten; Packer, Nicolle H.; Schulz, Benjamin L.

Molecular & Cellular Proteomics (2016), 15 (6), 1773-1790CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

A review. The glycoproteome remains severely understudied because of significant anal. challenges assocd. with glycoproteomics, the system-wide anal. of intact glycopeptides. This review introduces important structural aspects of protein N-glycosylation and summarizes the latest technol. developments and applications in LC-MS/MS-based qual. and quant. N-glycoproteomics. These maturing technologies provide unique structural insights into the N-glycoproteome and its synthesis and regulation by complementing existing methods in glycoscience. Modern glycoproteomics is now sufficiently mature to initiate efforts to capture the mol. complexity displayed by the N-glycoproteome, opening exciting opportunities to increase our understanding of the functional roles of protein N-glycosylation in human health and disease.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XovFWlsLk%253D&md5=867312c88588f344de22aebe46c489d3

106

Nilsson, J. Glycoconjugate J. 2016 , 33 , 261 – 272 DOI: 10.1007/s10719-016-9649-3

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106

Liquid chromatography-tandem mass spectrometry-based fragmentation analysis of glycopeptides

Nilsson, Jonas

Glycoconjugate Journal (2016), 33 (3), 261-272CODEN: GLJOEW; ISSN:0282-0080. (Springer)

The use of liq. chromatog.-electrospray ionization-tandem mass spectrometry (LC-ESI-MSn) for the glycoproteomic characterization of glycopeptides is a growing field of research. The N- and O-glycosylated peptides (N- and O-glycopeptides) analyzed typically originate from protease-digested glycoproteins where many of them are expected to be biomedically important. Examples of LC-MS2 and MS3 fragmentation strategies used to pursue glycan structure, peptide identity and attachment-site identification analyses of glycopeptides are described in this review. MS2 spectra, using the CID and HCD fragmentation techniques of a complex biantennary N-glycopeptide and a core 1 O-glycopeptide, representing two examples of commonly studied glycopeptide types, are presented. A few practical tips for accomplishing glycopeptide anal. using reversed-phase LC-MSn shotgun proteomics settings, together with refs. to the latest glycoproteomic studies, are presented.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xps1Wqtg%253D%253D&md5=10afdb9c5be4828903821b65da35a390

107

Leymarie, N. ; Zaia, J. Anal. Chem. 2012 , 84 , 3040 – 3048 DOI: 10.1021/ac3000573

108

Porras-Yakushi, T. R. ; Sweredoski, M. J. ; Hess, S. J. Am. Soc. Mass Spectrom. 2015 , 26 , 1580 – 1587 DOI: 10.1007/s13361-015-1168-0

109

Crowe, S. O. ; Rana, A. S. J. B. ; Deol, K. K. ; Ge, Y. ; Strieter, E. R. Anal. Chem. 2017 , 89 , 4428 – 4434 DOI: 10.1021/acs.analchem.6b03675

110

Rana, A. S. J. B. ; Ge, Y. ; Strieter, E. R. J. Proteome Res. 2017 , 16 , 3363 – 3369 DOI: 10.1021/acs.jproteome.7b00381

[ACS Full Text ], [CAS], Google Scholar

110

Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) Reveals Cell-Cycle Dependent Formation of Lys11/Lys48 Branched Ubiquitin Chains

Rana, Ambar S. J. B.; Ge, Ying; Strieter, Eric R.

Journal of Proteome Research (2017), 16 (9), 3363-3369CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

The dynamics of cellular signaling events are tightly regulated by a diverse set of ubiquitin chains. Recent work has suggested that branched ubiquitin chains composed of Lys-11 and Lys-48 isopeptide linkages play a crit. role in regulating cell cycle progression. Yet, endogenous Lys-11/Lys-48 branched chains could not be detected. By combining a Lys-11 linkage-specific antibody with high-resoln. middle-down mass spectrometry (an approach termed UbiChEM-MS), we sought to identify endogenous Lys-11/Lys-48 branched ubiquitin chains in cells. Using asynchronous cells, we found that Lys-11-linked branched chains could only be detected upon co-treatment with a proteasome and non-selective deubiquitinase inhibitor. Releasing cells from mitotic arrest resulted in a marked accumulation of Lys-11/Lys-48 branched chains in which branch points represented ∼3-4% of the total ubiquitin population. This report highlighted the utility of UbiChEM-MS in characterizing the architecture of Lys-11 Ub chains under various cellular conditions and corroborates the formation of Lys-11/Lys-48 branched chains during mitosis.

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111

Daniels, C. M. ; Ong, S.-E. ; Leung, A. K. L. Methods in Molecular Biology 2017 , 1608 , 79 – 93 DOI: 10.1007/978-1-4939-6993-7_7

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111

ADP-Ribosylated Peptide Enrichment and Site Identification: The Phosphodiesterase-Based Method

Daniels Casey M; Ong Shao-En; Leung Anthony K L; Leung Anthony K L

Methods in molecular biology (Clifton, N.J.) (2017), 1608 (), 79-93 ISSN:.

Protein ADP-ribosylation is a posttranslational modification (PTM) that plays an important role in all major cellular processes, including DNA repair, cellular signaling, and RNA metabolism. Site identification for this PTM has recently become possible through the development of several mass spectrometry-based methods, a critical step in understanding the regulatory role played by mono(ADP-ribose) (MAR), poly(ADP-ribose) (PAR), and the enzymes which make these modifications: poly(ADP-ribose) polymerases (PARPs), best known for their role in DNA repair and as targets for chemotherapeutic PARP inhibitors. Here, we have described our method for enriching and identifying ADP-ribosylation events through the use of a phosphodiesterase to digest protein-conjugated ADP-ribose down to its attachment structure, phosphoribose. We also include here a guide to choosing between collision-induced dissociation (CID)-, higher-energy collisional dissociation (HCD)-, and electron-transfer dissociation (ETD)-based peptide fragmentation for the identification of phosphoribosylated peptides.

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Rosenthal, F. ; Nanni, P. ; Barkow-Oesterreicher, S. ; Hottiger, M. O. J. Proteome Res. 2015 , 14 , 4072 – 4079 DOI: 10.1021/acs.jproteome.5b00432

[ACS Full Text ], [CAS], Google Scholar

112

Optimization of LTQ-Orbitrap Mass Spectrometer Parameters for the Identification of ADP-Ribosylation Sites

Rosenthal, Florian; Nanni, Paolo; Barkow-Oesterreicher, Simon; Hottiger, Michael O.

Journal of Proteome Research (2015), 14 (9), 4072-4079CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

ADP-ribosylation of proteins alters their function or provides a scaffold for the recruitment of other proteins, thereby regulating several important cellular processes. Mono- or poly-ADP-ribosylation is catalyzed by different ADP-ribosyltransferases (ARTs) that have different subcellular localizations and modify different amino acid acceptor sites. However, our knowledge of ADP-ribosylated proteins and their acceptor amino acids is still limited due to the lack of suitable mass spectrometry (MS) tools. Here, we describe an MS approach for the detection of ADP-ribosylated peptides and identification of the ADP-ribose acceptor sites, combining higher-energy collisional dissocn. (HCD) and electron-transfer dissocn. (ETD) on an LTQ-Orbitrap mass spectrometer. The presence of diagnostic ions of ADP-ribose in the HCD spectra allowed us to detect putative ADP-ribosylated peptides to target in a second LC-MS/MS anal. The combination of HCD with ETD fragmentation gave a more comprehensive coverage of ADP-ribosylation sites than that with HCD alone. We successfully identified different ADP-ribose acceptor sites on several in vitro modified proteins. The combination of optimized HCD and ETD methods may be applied to complex samples, allowing comprehensive identification of ADP-ribosylation acceptor sites.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1agsrfM&md5=08238e41a3169a58961aeaffac8e8a8a

113

Bilan, V. ; Leutert, M. ; Nanni, P. ; Panse, C. ; Hottiger, M. O. Anal. Chem. 2017 , 89 , 1523 – 1530 DOI: 10.1021/acs.analchem.6b03365

114

Erce, M. A. ; Abeygunawardena, D. ; Low, J. K. K. ; Hart-Smith, G. ; Wilkins, M. R. Mol. Cell. Proteomics 2013 , 12 , 3184 – 3198 DOI: 10.1074/mcp.M113.031500

[Crossref], [PubMed], [CAS], Google Scholar

114

Interactions Affected by Arginine Methylation in the Yeast Protein-Protein Interaction Network

Erce, Melissa A.; Abeygunawardena, Dhanushi; Low, Jason K. K.; Hart-Smith, Gene; Wilkins, Marc R.

Molecular & Cellular Proteomics (2013), 12 (11), 3184-3198CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Protein-protein interactions can be modulated by the methylation of arginine residues. As a means of testing this, the authors recently described a conditional two-hybrid system, based on the bacterial adenylate cyclase (BACTH) system. Here, the authors used this conditional two-hybrid system to explore the effect of arginine methylation in modulating protein-protein interactions in a subset of the Saccharomyces cerevisiae arginine methylproteome network. Interactions between the yeast hub protein Npl3 and yeast proteins Air2, Ded1, Gbp2, Snp1, and Yra1 were first validated in the absence of methylation. The major yeast arginine methyltransferase Hmt1 was subsequently included in the conditional two-hybrid assay, initially to det. the degree of methylation that occurs. Proteins Snp1 and Yra1 were confirmed as Hmt1 substrates, with five and two novel arginine methylation sites mapped by ETD LC-MS/MS on these proteins, resp. Proteins Ded1 and Gbp2, previously predicted but not confirmed as substrates of Hmt1, also are methylated with five and seven sites mapped, resp. Air2 is a novel substrate of Hmt1 with two sites mapped. Finally, the authors studied the interactions of Npl3 with the five interaction partners in the presence of active Hmt1 and in the presence of Hmt1 with a G68R inactivation mutation. The interaction between Npl3 and Air2, and Npl3 and Ded1, were significantly increased in the presence of active Hmt1; the interaction of Npl3 and Snp1 showed a similar degree of increase in interaction but this was not statistically significant. The interactions of Npl3 and Gbp2, along with Npl3 and Yra1, were not significantly increased or decreased by methylation. Methylarginine may be a widespread means by which the interactions of proteins are modulated.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhslWhtLzF&md5=8e244fd97df3f8f146251988564ee639

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Gui, S. ; Gathiaka, S. ; Li, J. ; Qu, J. ; Acevedo, O. ; Hevel, J. M. J. Biol. Chem. 2014 , 289 , 9320 – 9327 DOI: 10.1074/jbc.M113.535278

116

Low, J. K. K. ; Im, H. ; Erce, M. A. ; Hart-Smith, G. ; Snyder, M. P. ; Wilkins, M. R. Proteomics 2016 , 16 , 465 – 476 DOI: 10.1002/pmic.201400564

117

Snijders, A. P. ; Hautbergue, G. M. ; Bloom, A. ; Williamson, J. C. ; Minshull, T. C. ; Phillips, H. L. ; Mihaylov, S. R. ; Gjerde, D. T. ; Hornby, D. P. ; Wilson, S. A. ; Hurd, P. J. ; Dickman, M. J. RNA 2015 , 21 , 347 – 359 DOI: 10.1261/rna.045138.114

118

Fisk, J. C. ; Li, J. ; Wang, H. ; Aletta, J. M. ; Qu, J. ; Read, L. K. Mol. Cell. Proteomics 2013 , 12 , 302 – 311 DOI: 10.1074/mcp.M112.022533

[Crossref], [PubMed], [CAS], Google Scholar

118

Proteomic analysis reveals diverse classes of arginine methylproteins in mitochondria of trypanosomes

Fisk, John C.; Li, Jun; Wang, Hao; Aletta, John M.; Qu, Jun; Read, Laurie K.

Molecular & Cellular Proteomics (2013), 12 (2), 302-311CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Arginine (arg) methylation is a widespread posttranslational modification of proteins that impacts numerous cellular processes such as chromatin remodeling, RNA processing, DNA repair, and cell signaling. Known arg methylproteins arise mostly from yeast and mammals, and are almost exclusively nuclear and cytoplasmic. Trypanosoma brucei is an early branching eukaryote whose genome encodes five putative protein arg methyltransferases, and thus likely contains a plethora of arg methylproteins. Addnl., trypanosomes and related organisms possess a unique mitochondrion that undergoes dramatic developmental regulation and uses novel RNA editing and mitochondrial DNA replication mechanisms. Here, we performed a global mass spectrometric anal. of the T. brucei mitochondrion to identify new arg methylproteins in this medically relevant parasite. Enabling factors of this work are use of a combination digestion with two orthogonal enzymes, an efficient offline two dimensional chromatog. sepn., and high-resoln. mass spectrometry anal. with two complementary activations. This approach led to the comprehensive, sensitive and confident identification and localization of methylarg at a proteome level. We identified 167 arg methylproteins with wide-ranging functions including metab., transport, chaperoning, RNA processing, translation, and DNA replication. Our data suggest that arg methylproteins in trypanosome mitochondria possess both trypanosome-specific and evolutionarily conserved modifications, depending on the protein targeted. This study is the first comprehensive anal. of mitochondrial arg methylation in any organism, and represents a significant advance in our knowledge of the range of arg methylproteins and their sites of modification. Moreover, these studies establish T. brucei as a model organism for the study of posttranslational modifications.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXitFert7o%253D&md5=e8af9939ad9be94e99888587c39078e3

119

Matheron, L. ; van den Toorn, H. ; Heck, A. J. R. ; Mohammed, S. Anal. Chem. 2014 , 86 , 8312 – 8320 DOI: 10.1021/ac501803z

120

de Graaf, E. L. ; Giansanti, P. ; Altelaar, A. F. M. ; Heck, A. J. R. Mol. Cell. Proteomics 2014 , 13 , 2426 – 2434 DOI: 10.1074/mcp.O113.036608

[Crossref], [PubMed], [CAS], Google Scholar

120

Single-step Enrichment by Ti4+-IMAC and Label-free Quantitation Enables In-depth Monitoring of Phosphorylation Dynamics with High Reproducibility and Temporal Resolution

de Graaf, Erik L.; Giansanti, Piero; Maarten Altelaar, A. F.; Heck, Albert J. R.

Molecular & Cellular Proteomics (2014), 13 (9), 2426-2434CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Quant. phosphoproteomics workflows traditionally involve addnl. sample labeling and fractionation steps for accurate and in-depth anal. Here we report a high-throughput, straightforward, and comprehensive label-free phosphoproteomics approach using the highly selective, reproducible, and sensitive Ti4+-IMAC phosphopeptide enrichment method. We demonstrate the applicability of this approach by monitoring the phosphoproteome dynamics of Jurkat T cells stimulated by prostaglandin E2 (PGE2) over six different time points, measuring in total 108 snapshots of the phosphoproteome. In total, we quant. monitored 12,799 unique phosphosites over all time points with very high quant. reproducibility (av. r > 0.9 over 100 measurements and a median cv < 0.2). PGE2 is known to increase cellular cAMP levels, thereby activating PKA. The in-depth anal. revealed temporal regulation of a wide variety of phosphosites assocd. not only with PKA, but also with a variety of other classes of kinases. Following PGE2 stimulation, several pathways became only transiently activated, revealing that in-depth dynamic profiling requires techniques with high temporal resoln. Moreover, the large publicly available dataset provides a valuable resource for downstream PGE2 signaling dynamics in T cells, and cAMP-mediated signaling in particular. More generally, our method enables in-depth, quant., high-throughput phosphoproteome screening on any system, requiring very little sample, sample prepn., and anal. time.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVynurrJ&md5=6942c531aae132ba9d907d7cd3b17592

121

Rayapuram, N. ; Bonhomme, L. ; Bigeard, J. ; Haddadou, K. ; Przybylski, C. ; Hirt, H. ; Pflieger, D. J. Proteome Res. 2014 , 13 , 2137 – 2151 DOI: 10.1021/pr401268v

[ACS Full Text ], [CAS], Google Scholar

121

Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in Arabidopsis thaliana by quantitative phosphoproteomic analysis

Rayapuram, Naganand; Bonhomme, Ludovic; Bigeard, Jean; Haddadou, Kahina; Przybylski, Cedric; Hirt, Heribert; Pflieger, Delphine

Journal of Proteome Research (2014), 13 (4), 2137-2151CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-assocd. mol. pattern (PAMP), the authors searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissocn. (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, they identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible.

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Giansanti, P. ; Aye, T. T. ; van den Toorn, H. ; Peng, M. ; van Breukelen, B. ; Heck, A. J. R. Cell Rep. 2015 , 11 , 1834 – 1843 DOI: 10.1016/j.celrep.2015.05.029

[Crossref], [PubMed], [CAS], Google Scholar

122

An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas

Giansanti, Piero; Aye, Thin Thin; van den Toorn, Henk; Peng, Mao; van Breukelen, Bas; Heck, Albert J. R.

Cell Reports (2015), 11 (11), 1834-1843CODEN: CREED8; ISSN:2211-1247. (Cell Press)

Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti4+-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS). For specific sites, this approach increases their detectability by more than 1,000-fold.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVeiu7rN&md5=498359b688d194589be6698c996fdb11

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Joshi, R. N. ; Binai, N. A. ; Marabita, F. ; Sui, Z. ; Altman, A. ; Heck, A. J. R. ; Tegnér, J. ; Schmidt, A. Front. Immunol. 2017 , 8 , 1163 DOI: 10.3389/fimmu.2017.01163

[Crossref], [PubMed], [CAS], Google Scholar

123

Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

Joshi Rubin N; Marabita Francesco; Tegner Jesper; Schmidt Angelika; Binai Nadine A; Heck Albert J R; Binai Nadine A; Heck Albert J R; Sui Zhenhua; Altman Amnon; Tegner Jesper; Tegner Jesper

Frontiers in immunology (2017), 8 (), 1163 ISSN:1664-3224.

Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4(+)CD25(-) T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP3 receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M%252FmvVOqug%253D%253D&md5=23cadf8f9ddaee26ad3b89f78c57526f

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Bailey, D. J. ; Rose, C. M. ; McAlister, G. C. ; Brumbaugh, J. ; Yu, P. ; Wenger, C. D. ; Westphall, M. S. ; Thomson, J. A. ; Coon, J. J. Proc. Natl. Acad. Sci. U. S. A. 2012 , 109 , 8411 – 8416 DOI: 10.1073/pnas.1205292109

125

Collins, M. O. ; Wright, J. C. ; Jones, M. ; Rayner, J. C. ; Choudhary, J. S. J. Proteomics 2014 , 103 , 1 – 14 DOI: 10.1016/j.jprot.2014.03.010

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125

Confident and sensitive phosphoproteomics using combinations of collision induced dissociation and electron transfer dissociation

Collins, Mark O.; Wright, James C.; Jones, Matthew; Rayner, Julian C.; Choudhary, Jyoti S.

Journal of Proteomics (2014), 103 (), 1-14CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)

We present a workflow using an ETD-optimized version of Mascot Percolator and a modified version of SLoMo (turbo-SLoMo) for anal. of phosphoproteomic data. We have benchmarked this against several database searching algorithms and phosphorylation site localisation tools and show that it offers highly sensitive and confident phosphopeptide identification and site assignment with PSM-level statistics, enabling rigorous comparison of data acquisition methods. We analyzed the Plasmodium falciparum schizont phosphoproteome using for the first time, a data-dependent neutral loss-triggered-ETD (DDNL) strategy and a conventional decision-tree method. At a posterior error probability threshold of 0.01, similar nos. of PSMs were identified using both methods with a 73% overlap in phosphopeptide identifications. The false discovery rate assocd. with spectral pairs where DDNL CID/ETD identified the same phosphopeptide was < 1%. 72% of phosphorylation site assignments using turbo-SLoMo without any score filtering, were identical and 99.8% of these cases are assocd. with a false localisation rate of < 5%. We show that DDNL acquisition is a useful approach for phosphoproteomics and results in an increased confidence in phosphopeptide identification without compromising sensitivity or duty cycle. Furthermore, the combination of Mascot Percolator and turbo-SLoMo represents a robust workflow for phosphoproteomic data anal. using CID and ETD fragmentation. Protein phosphorylation is a ubiquitous post-translational modification that regulates protein function. Mass spectrometry-based approaches have revolutionised its anal. on a large-scale but phosphorylation sites are often identified by single phosphopeptides and therefore require more rigorous data anal. to unsure that sites are identified with high confidence for follow-up expts. to investigate their biol. significance. The coverage and confidence of phosphoproteomic expts. can be enhanced by the use of multiple complementary fragmentation methods. Here we have benchmarked a data anal. pipeline for anal. of phosphoproteomic data generated using CID and ETD fragmentation and used it to demonstrate the utility of a data-dependent neutral loss triggered ETD fragmentation strategy for high confidence phosphopeptide identification and phosphorylation site localisation.

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Wiese, H. ; Kuhlmann, K. ; Wiese, S. ; Stoepel, N. S. ; Pawlas, M. ; Meyer, H. E. ; Stephan, C. ; Eisenacher, M. ; Drepper, F. ; Warscheid, B. J. Proteome Res. 2014 , 13 , 1128 – 1137 DOI: 10.1021/pr400402s

[ACS Full Text ], [CAS], Google Scholar

126

Comparison of Alternative MS/MS and Bioinformatics Approaches for Confident Phosphorylation Site Localization

Wiese, Heike; Kuhlmann, Katja; Wiese, Sebastian; Stoepel, Nadine S.; Pawlas, Magdalena; Meyer, Helmut E.; Stephan, Christian; Eisenacher, Martin; Drepper, Friedel; Warscheid, Bettina

Journal of Proteome Research (2014), 13 (2), 1128-1137CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Over the past years, phosphoproteomics has advanced to a prime tool in signaling research. Since then, an enormous amt. of information about in vivo protein phosphorylation events has been collected providing a treasure trove for gaining a better understanding of the mol. processes involved in cell signaling. Yet, we still face the problem of how to achieve correct modification site localization. Here we use alternative fragmentation and different bioinformatics approaches for the identification and confident localization of phosphorylation sites. Phosphopeptide-enriched fractions were analyzed by multistage activation, collision-induced dissocn. and electron transfer dissocn. (ETD), yielding complementary phosphopeptide identifications. We further found that MASCOT, OMSSA and Andromeda each identified a distinct set of phosphopeptides allowing the no. of site assignments to be increased. The postsearch engine SLoMo provided confident phosphorylation site localization, whereas different versions of PTM-Score integrated in MaxQuant differed in performance. Based on high-resoln. ETD and higher collisional dissocn. (HCD) data sets from a large synthetic peptide and phosphopeptide ref. library reported by Marx et al., we show that an Andromeda/PTM-Score probability of 1 is required to provide an false localization rate (FLR) of 1% for HCD data, while 0.55 is sufficient for high-resoln. ETD spectra. Addnl. analyses of HCD data demonstrated that for phosphotyrosine peptides and phosphopeptides contg. two potential phosphorylation sites, PTM-Score probability cutoff values of <1 can be applied to ensure an FLR of 1%. Proper adjustment of localization probability cutoffs allowed us to significantly increase the no. of confident sites with an FLR of <1%.Our findings underscore the need for the systematic assessment of FLRs for different score values to report confident modification site localization.

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Marx, H. ; Lemeer, S. ; Schliep, J. E. ; Matheron, L. ; Mohammed, S. ; Cox, J. ; Mann, M. ; Heck, A. J. R. ; Kuster, B. Nat. Biotechnol. 2013 , 31 , 557 – 564 DOI: 10.1038/nbt.2585

128

Penkert, M. ; Yates, L. M. ; Schümann, M. ; Perlman, D. ; Fiedler, D. ; Krause, E. Anal. Chem. 2017 , 89 , 3672 – 3680 DOI: 10.1021/acs.analchem.6b05095

129

Frese, C. K. ; Zhou, H. ; Taus, T. ; Altelaar, A. F. M. ; Mechtler, K. ; Heck, A. J. R. ; Mohammed, S. J. Proteome Res. 2013 , 12 , 1520 – 1525 DOI: 10.1021/pr301130k

[ACS Full Text ], [CAS], Google Scholar

129

Unambiguous Phosphosite Localization using Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)

Frese, Christian K.; Zhou, Houjiang; Taus, Thomas; Altelaar, A. F. Maarten; Mechtler, Karl; Heck, Albert J. R.; Mohammed, Shabaz

Journal of Proteome Research (2013), 12 (3), 1520-1525CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

We recently introduced a novel scheme combining electron-transfer and higher-energy collision dissocn. (termed EThcD), for improved peptide ion fragmentation and identification. We reasoned that phosphosite localization, one of the major hurdles in high-throughput phosphoproteomics, could also highly benefit from the generation of such EThcD spectra. Here, we systematically assessed the impact on phosphosite localization utilizing EThcD in comparison to methods employing either ETD or HCD, resp., using a defined synthetic phosphopeptide mixt. and also using a larger data set of Ti4+-IMAC enriched phosphopeptides from a tryptic human cell line digest. In combination with a modified version of phosphoRS, we obsd. that in the majority of cases EThcD generated richer and more confidently identified spectra, resulting in superior phosphosite localization scores. Our data demonstrates the distinctive potential of EThcD for PTM localization, also beyond protein phosphorylation.

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Taus, T. ; Köcher, T. ; Pichler, P. ; Paschke, C. ; Schmidt, A. ; Henrich, C. ; Mechtler, K. J. Proteome Res. 2011 , 10 , 5354 – 5362 DOI: 10.1021/pr200611n

[ACS Full Text ], [CAS], Google Scholar

130

Universal and Confident Phosphorylation Site Localization Using phosphoRS

Taus, Thomas; Koecher, Thomas; Pichler, Peter; Paschke, Carmen; Schmidt, Andreas; Henrich, Christoph; Mechtler, Karl

Journal of Proteome Research (2011), 10 (12), 5354-5362CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

An algorithm for the assignment of phosphorylation sites in peptides is described. The program uses tandem mass spectrometry data in conjunction with the resp. peptide sequences to calc. site probabilities for all potential phosphorylation sites. Tandem mass spectra from synthetic phosphopeptides were used for optimization of the scoring parameters employing all commonly used fragmentation techniques. Calcn. of probabilities was adapted to the different fragmentation methods and to the max. mass deviation of the anal. The software includes a novel approach to peak extn., required for matching exptl. data to the theor. values of all isoforms, by defining individual peak depths for the different regions of the tandem mass spectrum. Mixts. of synthetic phosphopeptides were used to validate the program by calcn. of its false localization rate vs. site probability cutoff characteristic. Notably, the empirical obtained precision was higher than indicated by the applied probability cutoff. In addn., the performance of the algorithm was compared to existing approaches to site localization such as Ascore. To assess the practical applicability of the algorithm to large data sets, phosphopeptides from a biol. sample were analyzed, localizing more than 3000 nonredundant phosphorylation sites. Finally, the results obtained for the different fragmentation methods and localization tools were compared and discussed.

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Lössl, P. ; Brunner, A. M. ; Liu, F. ; Leney, A. C. ; Yamashita, M. ; Scheltema, R. A. ; Heck, A. J. R. ACS Cent. Sci. 2016 , 2 , 445 – 455 DOI: 10.1021/acscentsci.6b00053

[ACS Full Text ], [CAS], Google Scholar

131

Deciphering the Interplay among Multisite Phosphorylation, Interaction Dynamics, and Conformational Transitions in a Tripartite Protein System

Lossl Philip; Brunner Andrea M; Liu Fan; Leney Aneika C; Scheltema Richard A; Heck Albert J R; Yamashita Masami

ACS central science (2016), 2 (7), 445-55 ISSN:2374-7943.

Multisite phosphorylation is a common pathway to regulate protein function, activity, and interaction pattern in vivo, but routine biochemical analysis is often insufficient to identify the number and order of individual phosphorylation reactions and their mechanistic impact on the protein behavior. Here, we integrate complementary mass spectrometry (MS)-based approaches to characterize a multisite phosphorylation-regulated protein system comprising Polo-like kinase 1 (Plk1) and its coactivators Aurora kinase A (Aur-A) and Bora, the interplay of which is essential for mitotic entry after DNA damage-induced cell cycle arrest. Native MS and cross-linking-MS revealed that Aur-A/Bora-mediated Plk1 activation is accompanied by the formation of Aur-A/Bora and Plk1/Bora heterodimers. We found that the Aur-A/Bora interaction is independent of the Bora phosphorylation state, whereas the Plk1/Bora interaction is dependent on extensive Bora multisite phosphorylation. Bottom-up and top-down proteomics analyses showed that Bora multisite phosphorylation proceeds via a well-ordered sequence of site-specific phosphorylation reactions, whereby we could reveal the involvement of up to 16 phosphorylated Bora residues. Ion mobility spectrometry-MS demonstrated that this multisite phosphorylation primes a substantial structural rearrangement of Bora, explaining the interdependence between extensive Bora multisite phosphorylation and Plk1/Bora complex formation. These results represent a first benchmark of our multipronged MS strategy, highlighting its potential to elucidate the mechanistic and structural implications of multisite protein phosphorylation.

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Tamara, S. ; Scheltema, R. A. ; Heck, A. J. R. ; Leney, A. C. Angew. Chem. 2017 , 129 , 13829 – 13832 DOI: 10.1002/ange.201706749

133

Schmidt, A. ; Ammerer, G. ; Mechtler, K. Proteomics 2013 , 13 , 945 – 954 DOI: 10.1002/pmic.201200240

134

Bertran-Vicente, J. ; Serwa, R. A. ; Schümann, M. ; Schmieder, P. ; Krause, E. ; Hackenberger, C. P. R. J. Am. Chem. Soc. 2014 , 136 , 13622 – 13628 DOI: 10.1021/ja507886s

135

Schmidt, A. ; Trentini, D. B. ; Spiess, S. ; Fuhrmann, J. ; Ammerer, G. ; Mechtler, K. ; Clausen, T. Mol. Cell. Proteomics 2014 , 13 , 537 – 550 DOI: 10.1074/mcp.M113.032292

[Crossref], [PubMed], [CAS], Google Scholar

135

Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response

Schmidt, Andreas; Trentini, Debora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

Molecular & Cellular Proteomics (2014), 13 (2), 537-550CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-pos. bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. The authors developed a mass spectrometric approach accounting for the peculiar chem. properties of phosphoarginine. The improved methodol. was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quant. anal. showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large no. of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.

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Bertran-Vicente, J. ; Schümann, M. ; Hackenberger, C. P. R. ; Krause, E. Anal. Chem. 2015 , 87 , 6990 – 6994 DOI: 10.1021/acs.analchem.5b01389

137

Trentini, D. B. ; Suskiewicz, M. J. ; Heuck, A. ; Kurzbauer, R. ; Deszcz, L. ; Mechtler, K. ; Clausen, T. Nature 2016 , 539 , 48 – 53 DOI: 10.1038/nature20122

138

Scott, N. E. ; Parker, B. L. ; Connolly, A. M. ; Paulech, J. ; Edwards, A. V. G. ; Crossett, B. ; Falconer, L. ; Kolarich, D. ; Djordjevic, S. P. ; Højrup, P. ; Packer, N. H. ; Larsen, M. R. ; Cordwell, S. J. Mol. Cell. Proteomics 2011 , 10 , M000031-MCP201 DOI: 10.1074/mcp.M000031-MCP201

[Crossref], [PubMed], [CAS], Google Scholar

138

Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, higher energy collisional dissociation, and electron transfer dissociation MS applied to the N-linked glycoproteome of Campylobacter jejuni

Scott Nichollas E; Parker Benjamin L; Connolly Angela M; Paulech Jana; Edwards Alistair V G; Crossett Ben; Falconer Linda; Kolarich Daniel; Djordjevic Steven P; Hojrup Peter; Packer Nicolle H; Larsen Martin R; Cordwell Stuart J

Molecular & cellular proteomics : MCP (2011), 10 (2), M000031-MCP201 ISSN:.

Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or β-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence.

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Ford, K. L. ; Zeng, W. ; Heazlewood, J. L. ; Bacic, A. Front. Plant Sci. 2015 , 6 , 674 DOI: 10.3389/fpls.2015.00674

[Crossref], [PubMed], [CAS], Google Scholar

139

Characterization of protein N-glycosylation by tandem mass spectrometry using complementary fragmentation techniques

Ford Kristina L; Zeng Wei; Bacic Antony; Heazlewood Joshua L

Frontiers in plant science (2015), 6 (), 674 ISSN:1664-462X.

The analysis of post-translational modifications (PTMs) by proteomics is regarded as a technically challenging undertaking. While in recent years approaches to examine and quantify protein phosphorylation have greatly improved, the analysis of many protein modifications, such as glycosylation, are still regarded as problematic. Limitations in the standard proteomics workflow, such as use of suboptimal peptide fragmentation methods, can significantly prevent the identification of glycopeptides. The current generation of tandem mass spectrometers has made available a variety of fragmentation options, many of which are becoming standard features on these instruments. We have used three common fragmentation techniques, namely CID, HCD, and ETD, to analyze a glycopeptide and highlight how an integrated fragmentation approach can be used to identify the modified residue and characterize the N-glycan on a peptide.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC283jsFGjsg%253D%253D&md5=8d35d776a3cb36e646bcbfb0d8402b32

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Williams, J. P. ; Pringle, S. ; Richardson, K. ; Gethings, L. ; Vissers, J. P. C. ; De Cecco, M. ; Houel, S. ; Chakraborty, A. B. ; Yu, Y. Q. ; Chen, W. ; Brown, J. M. Rapid Commun. Mass Spectrom. 2013 , 27 , 2383 – 2390 DOI: 10.1002/rcm.6684

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140

Characterization of glycoproteins using a quadrupole time-of-flight mass spectrometer configured for electron transfer dissociation

Williams, Jonathan P.; Pringle, Steven; Richardson, Keith; Gethings, Lee; Vissers, Johannes P. C.; De Cecco, Martin; Houel, Stephane; Chakraborty, Asish B.; Yu, Ying Qing; Chen, Weibin; Brown, Jeffery M.

Rapid Communications in Mass Spectrometry (2013), 27 (21), 2383-2390CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)

RATIONALE : Electron transfer dissocn. (ETD) within ion trapping mass spectrometers has proven to be a useful tool for the characterization of post-translational modifications. In this study, we describe the implementation of ETD upon a modified quadrupole time-of-flight (Q-ToF) system and methods for the anal. of glycoproteins. METHODS : Liq. chromatog. electrospray ionization mass spectrometry (LC/ESI-MS) was performed using a hybrid quadrupole/ion mobility/oa-ToF mass spectrometer equipped with ETD functionality. 1,4-Dicyanobenzene reagent anions necessary for the ETD reaction were generated from a glow discharge region located within the ESI source block. ETD reactions occurred in the stacked ring traveling wave ion guide (located after the quadrupole mass filter and prior to the oa-ToF mass analyzer). LC/ETD was performed upon 'super-charged' tryptic glycopeptide ions produced from the recombinant monoclonal antibody trastuzumab. LC/ETD was also performed on ions from the smaller glycopeptides obtained from erythropoietin. RESULTS : ETD performed upon the quadruply 'super-charged' N-linked glycopeptide ions of trastuzumab and the triply charged O-linked glycopeptide ions of erythropoietin provided both glycosylation site assignments and full sequence information, resp. Tandem mass (MS/MS) spectra employing collision-induced dissocn. (CID) were dominated by oxonium product ions hampering full peptide sequence characterization. CONCLUSIONS : LC/ETD on the Q-ToF system proved effective at characterizing a no. of different N-linked glyco-forms of the tryptic peptide, EEQYNSTYR, from trastuzumab as well as glyco-forms from the O-linked tryptic peptide, EASIPPDAASAAPLR, from erythropoietin. The data demonstrates that the glycopeptide site heterogeneity of trastuzumab and erythropoietin can be accurately characterized. In addn., the post-column mixing of the super-charging reagent, m-NBA, is an effective method to increase the precursor ion charge state and to improve ETD reaction efficiency.

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Barallobre-Barreiro, J. ; Baig, F. ; Fava, M. ; Yin, X. ; Mayr, M. J. Visualized Exp. 2017 , e55674 – e55674 DOI: 10.3791/55674

142

Parker, B. L. ; Thaysen-Andersen, M. ; Solis, N. ; Scott, N. E. ; Larsen, M. R. ; Graham, M. E. ; Packer, N. H. ; Cordwell, S. J. J. Proteome Res. 2013 , 12 , 5791 – 5800 DOI: 10.1021/pr400783j

[ACS Full Text ], [CAS], Google Scholar

142

Site-Specific Glycan-Peptide Analysis for Determination of N-Glycoproteome Heterogeneity

Parker, Benjamin L.; Thaysen-Andersen, Morten; Solis, Nestor; Scott, Nichollas E.; Larsen, Martin R.; Graham, Mark E.; Packer, Nicolle H.; Cordwell, Stuart J.

Journal of Proteome Research (2013), 12 (12), 5791-5800CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

A combined glycomics and glycoproteomics strategy was developed for the site-specific anal. of N-linked glycosylation heterogeneity from a complex mammalian protein mixt. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liq. chromatog.-tandem mass spectrometry (PGC-LC-MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liq. chromatog. (Zic-HILIC) and fractionated by reversed-phase liq. chromatog. (RPLC; pH 7.9). The resulting fractions were each sepd. into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compns. to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database. CID spectra were used to assign glycan structures identified in the glycomic anal. to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.

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Trinidad, J. C. ; Schoepfer, R. ; Burlingame, A. L. ; Medzihradszky, K. F. Mol. Cell. Proteomics 2013 , 12 , 3474 – 3488 DOI: 10.1074/mcp.M113.030007

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143

N- and O-Glycosylation in the Murine Synaptosome

Trinidad, Jonathan C.; Schoepfer, Ralf; Burlingame, Alma L.; Medzihradszky, Katalin F.

Molecular & Cellular Proteomics (2013), 12 (12), 3474-3488CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N- and O-linked carbohydrate structures. Liq. chromatog.-MS (LC/MS) anal. with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissocn. (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixt. Over 2500 unique N- and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFWku7bN&md5=49af9b464bb350dbd577da06510579e7

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Medzihradszky, K. F. ; Kaasik, K. ; Chalkley, R. J. Mol. Cell. Proteomics 2015 , 14 , 2103 – 2110 DOI: 10.1074/mcp.M115.050393

[Crossref], [PubMed], [CAS], Google Scholar

144

Tissue-Specific Glycosylation at the Glycopeptide Level

Medzihradszky, Katalin F.; Kaasik, Krista; Chalkley, Robert J.

Molecular & Cellular Proteomics (2015), 14 (8), 2103-2110CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

This manuscript describes the enrichment and mass spectrometric anal. of intact glycopeptides from mouse liver, which yielded site-specific N- and O-glycosylation data for ∼130 proteins. Incorporation of different sialic acid variants in both N- and O-linked glycans was obsd., and the importance of using both collisional activation and electron transfer dissocn. for glycopeptide anal. was illustrated. The N-glycan structures of predicted lysosomal, endoplasmic reticulum (ER), secreted and transmembrane proteins were compared. The data suggest that protein N-glycosylation differs depending on cellular location. The glycosylation patterns of several mouse liver and mouse brain glycopeptides were compared. Tissue-specific differences in glycosylation were obsd. between sites within the same protein: Some sites displayed a similar spectrum of glycan structures in both tissues, whereas for others no overlap was obsd. We present comparative brain/liver glycosylation data on 50 N-glycosylation sites from 34 proteins and 13 O-glycosylation sites from seven proteins.

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Xu, S.-L. ; Medzihradszky, K. F. ; Wang, Z.-Y. ; Burlingame, A. L. ; Chalkley, R. J. Mol. Cell. Proteomics 2016 , 15 , 2048 – 2054 DOI: 10.1074/mcp.M115.056101

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145

N-Glycopeptide Profiling in Arabidopsis Inflorescence

Xu, Shou-Ling; Medzihradszky, Katalin F.; Wang, Zhi-Yong; Burlingame, Alma L.; Chalkley, Robert J.

Molecular & Cellular Proteomics (2016), 15 (6), 2048-2054CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

This study presents the first large-scale anal. of plant intact glycopeptides. Using wheat germ agglutinin lectin weak affinity chromatog. to enrich modified peptides, followed by electron transfer dissocn. (ETD)1 fragmentation tandem mass spectrometry, glycan compns. on over 1100 glycopeptides from 270 proteins found in Arabidopsis inflorescence tissue were characterized. While some sites were only detected with a single glycan attached, others displayed up to 16 different glycoforms. Among the identified glycopeptides were four modified in nonconsensus glycosylation motifs. While most of the modified proteins are secreted, membrane, endoplasmic reticulum (ER), or Golgi-localized proteins, surprisingly, N-linked sugars were detected on a protein predicted to be cytosolic or nuclear.

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146

Darula, Z. ; Medzihradszky, K. F. J. Am. Soc. Mass Spectrom. 2014 , 25 , 977 – 987 DOI: 10.1007/s13361-014-0852-9

147

Saba, J. ; Dutta, S. ; Hemenway, E. ; Viner, R. Int. J. Proteomics 2012 , 2012 , 560391 DOI: 10.1155/2012/560391

[Crossref], [PubMed], [CAS], Google Scholar

147

Increasing the productivity of glycopeptides analysis by using higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation

Saba Julian; Dutta Sucharita; Hemenway Eric; Viner Rosa

International journal of proteomics (2012), 2012 (), 560391 ISSN:.

Currently, glycans are attracting attention from the scientific community as potential biomarkers or as posttranslational modifications (PTMs) of therapeutic proteins. However, structural characterization of glycoproteins and glycopeptides remains analytically challenging. Here, we report on the implementation of a novel acquisition strategy termed higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation (HCD-PD-ETD) on a hybrid linear ion trap-orbitrap mass spectrometer. This acquisition strategy uses the complementary fragmentations of ETD and HCD for glycopeptides analysis in an intelligent fashion. Furthermore, the approach minimizes user input for optimizing instrumental parameters and enables straightforward detection of glycopeptides. ETD spectra are only acquired when glycan oxonium ions from MS/MS HCD are detected. The advantage of this approach is that it streamlines data analysis and improves dynamic range and duty cycle. Here, we present the benefits of HCD-PD-ETD relative to the traditional alternating HCD/ETD for a trainer set containing twelve-protein mixture with two glycoproteins: human serotransferrin, ovalbumin and contaminations of two other: bovine alpha 1 acid glycoprotein (bAGP) and bovine fetuin.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38jitVejsA%253D%253D&md5=7051954e036d386ada67c2e0852a7a38

148

Singh, C. ; Zampronio, C. G. ; Creese, A. J. ; Cooper, H. J. J. Proteome Res. 2012 , 11 , 4517 – 4525 DOI: 10.1021/pr300257c

[ACS Full Text ], [CAS], Google Scholar

148

Higher energy collision dissociation (HCD) product ion-triggered electron transfer dissociation (ETD) mass spectrometry for the analysis of N-linked glycoproteins

Singh, Charandeep; Zampronio, Cleidiane G.; Creese, Andrew J.; Cooper, Helen J.

Journal of Proteome Research (2012), 11 (9), 4517-4525CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Large scale mass spectrometry anal. of N-linked glycopeptides is complicated by the inherent complexity of the glycan structures. Here, the authors evaluate a mass spectrometry approach for the targeted anal. of N-linked glycopeptides in complex mixts. that does not require prior knowledge of the glycan structures or pre-enrichment of the glycopeptides. Despite the complexity of N-glycans, the core of the glycan remains const., comprising two N-acetylglucosamine and three mannose units. Collision-induced dissocn. (CID) mass spectrometry of N-glycopeptides gave the N-acetylglucosamine (GlcNAc) oxonium ion and a [mannose+GlcNAc] fragment (in addn. to other fragments resulting from cleavage within the glycan). In ion-trap CID, those ions are not detected due to the low m/z cutoff; however, they were detected following the beam-type CID known as higher energy collision dissocn. (HCD) on the Orbitrap mass spectrometer. The presence of these product ions following HCD can be used as triggers for subsequent electron transfer dissocn. (ETD) mass spectrometry anal. of the precursor ion. The ETD mass spectrum provides peptide sequence information, which is unobtainable from HCD. A Lys-C digest of RNase B and trypsin digest of IgG were sepd. by ZIC-HILIC liq. chromatog. and analyzed by HCD product ion-triggered ETD. The data were analyzed both manually and by search against protein databases by commonly used algorithms. The product ion-triggered approach shows promise for the field of glycoproteomics and highlight the requirement for more sophisticated data mining tools.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVemur%252FP&md5=f54f61a701665e1f4bc5701fc627fc4d

149

Wu, S. W. ; Pu, T. H. ; Viner, R. ; Khoo, K. H. Anal. Chem. 2014 , 86 , 5478 – 5486 DOI: 10.1021/ac500945m

150

Levery, S. B. ; Steentoft, C. ; Halim, A. ; Narimatsu, Y. ; Clausen, H. ; Vakhrushev, S. Y. Biochim. Biophys. Acta, Gen. Subj. 2015 , 1850 , 33 – 42 DOI: 10.1016/j.bbagen.2014.09.026

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150

Advances in mass spectrometry driven O-glycoproteomics

Levery, Steven B.; Steentoft, Catharina; Halim, Adnan; Narimatsu, Yoshiki; Clausen, Henrik; Vakhrushev, Sergey Y.

Biochimica et Biophysica Acta, General Subjects (2015), 1850 (1), 33-42CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)

A review. Global analyses of proteins and their modifications by mass spectrometry are essential tools in cell biol. and biomedical research. Analyses of glycoproteins represent particular challenges and the authors are only at the beginnings of the glycoproteomic era. Some of the challenges have been overcome with N-glycoproteins and proteome-wide anal. of N-glycosylation sites is accomplishable today but only by sacrificing information of structures at individual glycosites. More recently advances in anal. of O-glycoproteins have been made and proteome-wide anal. of O-glycosylation sites is becoming available as well. Here the challenges of anal. of O-glycans and new O-glycoproteomics strategies focusing on O-GalNAc and O-Man glycoproteomes. A variety of strategies are now available for proteome-wide anal. of O-glycosylation sites enabling functional studies are discussed. However, further developments are still needed for complete anal. of glycan structures at individual sites for both N- and O-glycoproteomics strategies. The advances in O-glycoproteomics led to identification of new biol. functions of O-glycosylation and a new understanding of the importance of where O-glycans are positioned on proteins.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslagu7rE&md5=3059fae457a8d2f34aabb88a074405bb

151

Thaysen-Andersen, M. ; Wilkinson, B. L. ; Payne, R. J. ; Packer, R. H. Electrophoresis 2011 , 32 , 3536 – 3545 DOI: 10.1002/elps.201100294

[Crossref], [PubMed], [CAS], Google Scholar

151

Site-specific characterization of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS

Thaysen-Andersen, Morten; Wilkinson, Brendan L.; Payne, Richard J.; Packer, Nicolle H.

Electrophoresis (2011), 32 (24), 3536-3545CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)

Site-specific characterization of mucin-type O-linked glycosylation is an anal. challenge due to glycan heterogeneity, lack of glycosylation site consensus sequence and high d. of occupied glycosylation sites. Here, we report the use of electron transfer dissocn. (ETD) for the site-specific characterization of densely glycosylated mucin-type O-linked glycopeptides using ESI-IT-MS/MS. Synthetic glycopeptides from the human mucin-1 (MUC-1) tandem repeat region contg. a range of O-linked, tumor-assocd. carbohydrate antigens, namely Tn, T and sialyl T, with different glycosylation site occupancies and an increasing no. of tandem repeats were studied. In addn., a glycopeptide from the anti-freeze glycoprotein of Antarctic and Arctic notothenoids, bearing four O-linked, per-acetylated T antigens was characterized. ETD MS/MS of infused or capillary LC-sepd. glycopeptides provided broad peptide sequence coverage (c/z·-type fragment ions) with intact glycans still attached to the Ser/Thr residues. Thus, the glycosylation sites were unambiguously detd., while simultaneously obtaining information about the attached glycan mass and peptide identity. Highly sialylated O-glycopeptides showed less efficient peptide fragmentation, but some sequence and glycosylation site information was still obtained. This study demonstrates the capabilities of ETD MS/MS for site-specific characterization of mucin-type glycopeptides contg. high-d. O-linked glycan clusters, using accessible and relative low-resoln./low-mass accuracy IT MS instrumentation.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhs1aisbbK&md5=161b3a649ee27ce849f956eaaaa14a16

152

Darula, Z. ; Sherman, J. ; Medzihradszky, K. F. Mol. Cell. Proteomics 2012 , 11 , 1 – 10 DOI: 10.1074/mcp.O111.016774

153

Darula, Z. ; Sarnyai, F. ; Medzihradszky, K. F. Glycoconjugate J. 2016 , 33 , 435 – 445 DOI: 10.1007/s10719-015-9630-6

[Crossref], [PubMed], [CAS], Google Scholar

153

O-glycosylation sites identified from mucin core-1 type glycopeptides from human serum

Darula, Zsuzsanna; Sarnyai, Farkas; Medzihradszky, Katalin F.

Glycoconjugate Journal (2016), 33 (3), 435-445CODEN: GLJOEW; ISSN:0282-0080. (Springer)

In this work O-linked glycopeptides bearing mucin core-1 type structures were enriched from human serum. Since about 70 % of the O-glycans in human serum bind to the plant lectin Jacalin, we tested a previously successful protocol that combined Jacalin affinity enrichment on the protein- and peptide-level with ERLIC chromatog. as a further enrichment step in between, to eliminate the high background of unmodified peptides. In parallel, we developed a simpler and significantly faster new workflow that used two lectins sequentially: wheat germ agglutinin and then Jacalin. The first lectin provides general glycopeptide enrichment, while the second specifically enriches O-linked glycopeptides with Galβ1-3GalNAcα structures. Mass spectrometric anal. of enriched samples showed that the new sample prepn. method is more selective and sensitive than the former. Altogether, 52 unique glycosylation sites in 20 proteins were identified in this study.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xislaltg%253D%253D&md5=13d30091f5bed8b3e8c6347af6810b51

154

Hoffmann, M. ; Marx, K. ; Reichl, U. ; Wuhrer, M. ; Rapp, E. Mol. Cell. Proteomics 2016 , 15 , 624 – 641 DOI: 10.1074/mcp.M115.053546

[Crossref], [PubMed], [CAS], Google Scholar

154

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins

Hoffmann, Marcus; Marx, Kristina; Reichl, Udo; Wuhrer, Manfred; Rapp, Erdmann

Molecular & Cellular Proteomics (2016), 15 (2), 624-641CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Site-specific glycosylation anal. is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The anal., though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clin. and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation anal. of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a pptn. step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liq. chromatog., the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric anal. using reversed-phase liq. chromatog. coupled online to an ion trap mass spectrometer operated in pos.-ion mode. Peptide identity and glycan compn. were derived from low-energy collision-induced dissocn. fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissocn. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compns. most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslOktL8%253D&md5=9eec845955e5dcb7ba05d4320c49c34e

155

Houel, S. ; Hilliard, M. ; Yu, Y. Q. ; McLoughlin, N. ; Martin, S. M. ; Rudd, P. M. ; Williams, J. P. ; Chen, W. Anal. Chem. 2014 , 86 , 576 – 584 DOI: 10.1021/ac402726h

[ACS Full Text ], [CAS], Google Scholar

155

N- and O-Glycosylation Analysis of Etanercept Using Liquid Chromatography and Quadrupole Time-of-Flight Mass Spectrometry Equipped with Electron-Transfer Dissociation Functionality

Houel, Stephane; Hilliard, Mark; Yu, Ying Qing; McLoughlin, Niaobh; Martin, Silvia Millan; Rudd, Pauline M.; Williams, Jonathan P.; Chen, Weibin

Analytical Chemistry (Washington, DC, United States) (2014), 86 (1), 576-584CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Etanercept is a highly glycosylated therapeutic Fc-fusion protein that contains multiple N- and O-glycosylation sites. An in-depth characterization of the glycosylation of etanercept was carried out using liq. chromatog./mass spectrometry (LC/MS) methods in a systematic approach in which we analyzed the N- and O-linked glycans and located the occupied O-glycosylation sites. Etanercept was first treated with peptide N-glycosidase F to release the N-glycans. The N-glycan pool was labeled with a 2-aminobenzamide (2-AB) fluorescence tag and sepd. using ultraperformance liq. chromatog.-hydrophilic interaction liq. chromatog. (UPLC-HILIC). Preliminary structures were assigned using Glycobase. These assignments, which included monosaccharide sequence and linkage information, were confirmed by exoglycosidase array digestions of aliquots of the N-glycan pool. The removal of the N-glycans from etanercept facilitated the selective characterization of O-glycopeptides and enabled the O-glycans to be identified. These were predominantly of the core 1 subtype (HexHexNAc O-structure) attached to Ser/Thr residues. α2→3,6,8,9 sialidase was used to remove the sialic acid residues on the O-glycans allowing the use of an automated LC/MSE protocol to identify the O-glycopeptides. Electron-transfer dissocn. (ETD) was then used to pinpoint the 12 occupied O-glycosylation sites. The detn. of N- and O-glycans and O-glycosylation sites in etanercept provides a basis for future studies addressing the biol. importance of specific protein glycosylations in the prodn. of safe and efficacious biotherapeutics.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvV2rsrfF&md5=21a3b1b563166fcb27b4d5856934752a

156

Steentoft, C. ; Vakhrushev, S. Y. ; Vester-Christensen, M. B. ; Schjoldager, K. T. B. G. ; Kong, Y. ; Bennett, E. P. ; Mandel, U. ; Wandall, H. ; Levery, S. B. ; Clausen, H. Nat. Methods 2011 , 8 , 977 – 982 DOI: 10.1038/nmeth.1731

[Crossref], [PubMed], [CAS], Google Scholar

156

Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines

Steentoft, Catharina; Vakhrushev, Sergey Y.; Vester-Christensen, Malene B.; Schjoldager, Katrine T.-B. G.; Kong, Yun; Bennett, Eric Paul; Mandel, Ulla; Wandall, Hans; Levery, Steven B.; Clausen, Henrik

Nature Methods (2011), 8 (11), 977-982CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

Zinc-finger nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed anal. of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN targeting to truncate the O-glycan elongation pathway in human cells, generating stable 'SimpleCell' lines with homogenous O-glycosylation. Three SimpleCell lines expressing only truncated GalNAcα or NeuAcα2-6GalNAcα O-glycans were produced, allowing straightforward isolation and sequencing of GalNAc O-glycopeptides from total cell lysates using lectin chromatog. and nanoflow liq. chromatog.-mass spectrometry (nLC-MS/MS) with electron transfer dissocn. fragmentation. We identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O-glycan linkage to a tyrosine residue. The SimpleCell method should facilitate analyses of important functions of protein glycosylation. The strategy is also applicable to other O-glycoproteomes.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht12iu7rK&md5=da053d265e2f69191144ba83772a22bd

157

Steentoft, C. ; Vakhrushev, S. Y. ; Joshi, H. J. ; Kong, Y. ; Vester-Christensen, M. B. ; Schjoldager, K. T. B. G. ; Lavrsen, K. ; Dabelsteen, S. ; Pedersen, N. B. ; Marcos-Silva, L. ; Gupta, R. ; Paul Bennett, E. ; Mandel, U. ; Brunak, S. ; Wandall, H. H. ; Levery, S. B. ; Clausen, H. EMBO J. 2013 , 32 , 1478 – 1488 DOI: 10.1038/emboj.2013.79

[Crossref], [PubMed], [CAS], Google Scholar

157

Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology

Steentoft, Catharina; Vakhrushev, Sergey Y.; Joshi, Hiren J.; Kong, Yun; Vester-Christensen, Malene B.; Schjoldager, Katrine T-B. G.; Lavrsen, Kirstine; Dabelsteen, Sally; Pedersen, Nis B.; Marcos-Silva, Lara; Gupta, Ramneek; Paul Bennett, Eric; Mandel, Ulla; Brunak, Soren; Wandall, Hans H.; Levery, Steven B.; Clausen, Henrik

EMBO Journal (2013), 32 (10), 1478-1488CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc-type O-glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc-transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O-glycosylation (SimpleCells) that enables proteome-wide discovery of O-glycan sites using bottom-up' ETD-based mass spectrometric anal. We implemented this on 12 human cell lines from different organs, and present a first map of the human O-glycoproteome with almost 3000 glycosites in over 600 O-glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O-glycosylation. The finding of unique subsets of O-glycoproteins in each cell line provides evidence that the O-glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O-glycoproteome should facilitate the exploration of how site-specific O-glycosylation regulates protein function.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXlslyqsLw%253D&md5=59fe0a4aa1936c95f88579acb227b66c

158

Yang, Z. ; Halim, A. ; Narimatsu, Y. ; Joshi, H. J. ; Steentoft, C. ; Schjoldager, K. T. B. G. ; Schulz, M. A. ; Sealover, N. R. ; Kayser, K. J. ; Bennett, E. P. ; Levery, S. B. ; Vakhrushev, S. Y. ; Clausen, H. Mol. Cell. Proteomics 2014 , 13 , 3224 – 3235 DOI: 10.1074/mcp.M114.041541

[Crossref], [PubMed], [CAS], Google Scholar

158

The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

Yang, Zhang; Halim, Adnan; Narimatsu, Yoshiki; Joshi, Hiren Jitendra; Steentoft, Catharina; Ter-Borch Gram Schjoldager, Katrine; Schulz, Morten Alder; Sealover, Natalie R.; Kayser, Kevin J.; Bennett, Eric Paul; Levery, Steven B.; Vakhrushev, Sergey Y.; Clausen, Henrik

Molecular & Cellular Proteomics (2014), 13 (12), 3224-3235CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant prodn. of biol. therapeutics primarily because of its "human-like" glycosylation features. CHO is used for prodn. of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for prodn. of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We identified 738 O-glycoproteins (1548 O-glycosites) in cell lysates and secretomes providing the first comprehensive insight into the O-glycosylation capacity of CHO (http://glycomics.ku.dk/o-glycoproteome_db/).

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvF2rtrzK&md5=b575b647ae6d86b482019a959e0743ee

159

Neubert, P. ; Halim, A. ; Zauser, M. ; Essig, A. ; Joshi, H. J. ; Zatorska, E. ; Larsen, I. S. B. ; Loibl, M. ; Castells-Ballester, J. ; Aebi, M. ; Clausen, H. ; Strahl, S. Mol. Cell. Proteomics 2016 , 15 , 1323 – 1337 DOI: 10.1074/mcp.M115.057505

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159

Mapping the O-mannose glycoproteome in Saccharomyces cerevisiae

Neubert, Patrick; Halim, Adnan; Zauser, Martin; Essig, Andreas; Joshi, Hiren J.; Zatorska, Ewa; Bohse Larsen, Ida Signe; Loibl, Martin; Castells-Ballester, Joan; Aebi, Markus; Clausen, Henrik; Strahl, Sabine

Molecular & Cellular Proteomics (2016), 15 (4), 1323-1337CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

O-Mannosylation is a vital protein modification conserved from fungi to humans. Yeast is a perfect model to study this post-translational modification, because in contrast to mammals O-mannosylation is the only type of O-glycosylation. In an essential step toward the full understanding of protein O-mannosylation we mapped the O-mannose glycoproteome in baker's yeast. Taking advantage of an O-glycan elongation deficient yeast strain to simplify sample complexity, we identified over 500 O-glycoproteins from all subcellular compartments for which over 2300 O-mannosylation sites were mapped by electron-transfer dissocn. (ETD)-based MS/MS. In this study, we focus on the 293 O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized protein O-mannosyltransferases. We find that O-mannosylation is not only a prominent modification of cell wall and plasma membrane proteins, but also of a large no. of proteins from the secretory pathway with crucial functions in protein glycosylation, folding, quality control, and trafficking. The anal. of glycosylation sites revealed that O-mannosylation is favored in unstructured regions and β-strands. Furthermore, O-mannosylation is impeded in the proximity of N-glycosylation sites suggesting the interplay of these types of post-translational modifications. The detailed knowledge of the target proteins and their O-mannosylation sites opens for discovery of new roles of this essential modification in eukaryotes, and for a first glance on the evolution of different types of O-glycosylation from yeast to mammals.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xltlansb8%253D&md5=e338e3ad41c93690604ab8db46ec6a01

160

Windwarder, M. ; Altmann, F. J. Proteomics 2014 , 108 , 258 – 268 DOI: 10.1016/j.jprot.2014.05.022

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160

Site-specific analysis of the O-glycosylation of bovine fetuin by electron-transfer dissociation mass spectrometry

Windwarder, Markus; Altmann, Friedrich

Journal of Proteomics (2014), 108 (), 258-268CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)

Bovine fetuin often finds use as a test model for anal. methods, but the exact occupancy of its O-glycosylation sites has not yet been detd. An obstacle for a closer inspection of the five or six O-glycosylation sites is the close spacing of several sites on the same tryptic peptide. The advent of ion-trap instruments with electron-transfer dissocn. (ETD) capability and - for the type of instrument - high resoln. prompted us to probe this technol. for the investigation of the intricate posttranslational modifications O-glycosylation and phosphorylation. Much information could be obtained by direct-infusion ETD anal. of the fully sialylated tryptic 61-residue peptide harboring 8 hydroxyl amino acids of which four were indeed found to be, if only partially, glycosylated. The middle-down approach allowed recognizing an order of action of O-GalNAc transferase(s). No such hierarchy could be obsd. for phosphorylation. ETD fragmentation on an ion trap thus allowed in-depth anal. of a large, multiply O-glycosylated peptide, however, only by data accumulation over several minutes by direct infusion of a prefractionated sample. O-glycosylation and phosphorylation sites re-defined and their occupancy including that of N-glycans were defined by this study. O-glycosylation of natural or recombinant proteins poses a challenge because of the lack of unambiguous consensus sites, the agglomeration of several O-glycans in close proximity and the lack of efficient O-glycosidases. Even bovine fetuin, a frequently used test glycoprotein for glycosylation anal., has hitherto not been fully characterized in terms of site occupancy. This gap shall hereby be closed by application of electron-transfer dissocn. mass spectroscopy.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFyjtLnO&md5=ac594133a690370e45797987e7c3518f

161

Ma, J. ; Hart, G. W. Analysis of Protein O-GlcNAcylation by Mass Spectrometry Current Protocols in Protein Science 2017 , 2017 ( John Wiley & Sons, Inc.: Hoboken, NJ ) 24.10.1 – 24.10.16 DOI: 10.1002/cpps.24

162

Myers, S. A. ; Daou, S. ; Affar, E. B. ; Burlingame, A. Proteomics 2013 , 13 , 982 – 991 DOI: 10.1002/pmic.201200332

163

Leney, A. C. ; El Atmioui, D. ; Wu, W. ; Ovaa, H. ; Heck, A. J. R. Proc. Natl. Acad. Sci. U. S. A. 2017 , 114 , E7255 – E7261 DOI: 10.1073/pnas.1620529114

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163

Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation

Leney, Aneika C.; El Atmioui, Dris; Wu, Wei; Ovaa, Huib; Heck, Albert J. R.

Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (35), E7255-E7261CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

Proteins can be modified by multiple post-translational modifications (PTMs), creating a PTM code that controls the function of proteins in space and time. Unraveling this complex PTM code is one of the great challenges in mol. biol. Here, using mass spectrometry-based assays, we focused on the most common PTMs, phosphorylation and O-GlcNAcylation, and investigated how they affect each other. We demonstrated 2 generic crosstalk mechanisms. First, we defined a frequently occurring, very specific and stringent phosphorylation/O-GlcNAcylation interplay motif, (pSp/T)P(V/A/T)(gS/gT), whereby phosphorylation strongly inhibits O-GlcNAcylation. Strikingly, this stringent motif was substantially enriched in the human (phospho)proteome, allowing us to predict hundreds of putative O-GlcNAc transferase (OGT) substrates. A set of these we investigated further and showed them to be decent substrates of OGT, exhibiting a neg. feedback loop when phosphorylated at the P-3 site. Second, we demonstrate that reciprocal crosstalk does not occur at PX(S/T)P sites, i.e., at sites phosphorylated by proline-directed kinases, which represent 40% of all sites in the vertebrate phosphoproteomes.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlemu7rL&md5=29df73b1585f35e3e342192894ac5244

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Trinidad, J. C. ; Barkan, D. T. ; Gulledge, B. F. ; Thalhammer, A. ; Sali, A. ; Schoepfer, R. ; Burlingame, A. L. Mol. Cell. Proteomics 2012 , 11 , 215 – 229 DOI: 10.1074/mcp.O112.018366

[Crossref], [PubMed], [CAS], Google Scholar

164

Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse

Trinidad Jonathan C; Barkan David T; Gulledge Brittany F; Thalhammer Agnes; Sali Andrej; Schoepfer Ralf; Burlingame Alma L

Molecular & cellular proteomics : MCP (2012), 11 (8), 215-29 ISSN:.

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic, reversible monosaccharide modifier of serine and threonine residues on intracellular protein domains. Crosstalk between O-GlcNAcylation and phosphorylation has been hypothesized. Here, we identified over 1750 and 16,500 sites of O-GlcNAcylation and phosphorylation from murine synaptosomes, respectively. In total, 135 (7%) of all O-GlcNAcylation sites were also found to be sites of phosphorylation. Although many proteins were extensively phosphorylated and minimally O-GlcNAcylated, proteins found to be extensively O-GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the O-GlcNAcylation system is specifically targeting a subset of the proteome that is also phosphorylated. Both PTMs usually occur on disordered regions of protein structure, within which, the location of O-GlcNAcylation and phosphorylation is virtually random with respect to each other, suggesting that negative crosstalk at the structural level is not a common phenomenon. As a class, protein kinases are found to be more extensively O-GlcNAcylated than proteins in general, indicating the potential for crosstalk of phosphorylation with O-GlcNAcylation via regulation of enzymatic activity.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38nnt1OitQ%253D%253D&md5=371d7fa144215c521e402a40c73cce7a

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Morris, M. ; Knudsen, G. M. ; Maeda, S. ; Trinidad, J. C. ; Ioanoviciu, A. ; Burlingame, A. L. ; Mucke, L. Nat. Neurosci. 2015 , 18 , 1183 – 1189 DOI: 10.1038/nn.4067

[Crossref], [PubMed], [CAS], Google Scholar

165

Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice

Morris, Meaghan; Knudsen, Giselle M.; Maeda, Sumihiro; Trinidad, Jonathan C.; Ioanoviciu, Alexandra; Burlingame, Alma L.; Mucke, Lennart

Nature Neuroscience (2015), 18 (8), 1183-1189CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)

The microtubule-assocd. protein tau has been implicated in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative disorders. Reducing tau levels ameliorates AD-related synaptic, network, and behavioral abnormalities in transgenic mice expressing human amyloid precursor protein (hAPP). We used mass spectrometry to characterize the post-translational modification of endogenous tau isolated from wild-type and hAPP mice. We identified seven types of tau modifications at 63 sites in wild-type mice. Wild-type and hAPP mice had similar modifications, supporting the hypothesis that neuronal dysfunction in hAPP mice is enabled by physiol. forms of tau. Our findings provide clear evidence for acetylation and ubiquitination of the same lysine residues; some sites were also targeted by lysine methylation. Our findings refute the hypothesis of extensive O-linked N-acetylglucosamine (O-GlcNAc) modification of endogenous tau. The complex post-translational modification of physiol. tau suggests that tau is regulated by diverse mechanisms.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1WltLbK&md5=42c937b45046c6c8d9313447efef9103

166

Alfaro, J. F. ; Gong, C.-X. ; Monroe, M. E. ; Aldrich, J. T. ; Clauss, T. R. W. ; Purvine, S. O. ; Wang, Z. ; Camp, D. G. ; Shabanowitz, J. ; Stanley, P. ; Hart, G. W. ; Hunt, D. F. ; Yang, F. ; Smith, R. D. Proc. Natl. Acad. Sci. U. S. A. 2012 , 109 , 7280 – 7285 DOI: 10.1073/pnas.1200425109

167

Kaasik, K. ; Kivimäe, S. ; Allen, J. J. ; Chalkley, R. J. ; Huang, Y. ; Baer, K. ; Kissel, H. ; Burlingame, A. L. ; Shokat, K. M. ; Ptáček, L. J. ; Fu, Y.-H. Cell Metab. 2013 , 17 , 291 – 302 DOI: 10.1016/j.cmet.2012.12.017

[Crossref], [PubMed], [CAS], Google Scholar

167

Glucose Sensor O-GlcNAcylation Coordinates with Phosphorylation to Regulate Circadian Clock

Kaasik, Krista; Kivimae, Saul; Allen, Jasmina J.; Chalkley, Robert J.; Huang, Yong; Baer, Kristin; Kissel, Holger; Burlingame, Alma L.; Shokat, Kevan M.; Ptacek, Louis J.; Fu, Ying-Hui

Cell Metabolism (2013), 17 (2), 291-302CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)

Posttranslational modifications play central roles in myriad biol. pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a crit. factor in regulating complex GSK3β-dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3β. Interestingly, OGT activity is regulated by GSK3β; hence, OGT and GSK3β exhibit reciprocal regulation. Modulating O-GlcNAcylation levels alter circadian period length in both mice and Drosophila; conversely, protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addn., O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662-S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine-tune circadian clock.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXitFCit7o%253D&md5=dad0f69a1da905ebde454ee4b38f6dcb

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Bullen, J. W. ; Balsbaugh, J. L. ; Chanda, D. ; Shabanowitz, J. ; Hunt, D. F. ; Neumann, D. ; Hart, G. W. J. Biol. Chem. 2014 , 289 , 10592 – 10606 DOI: 10.1074/jbc.M113.523068

169

Yu, Q. ; Wang, B. ; Chen, Z. ; Urabe, G. ; Glover, M. S. ; Shi, X. ; Guo, L. W. ; Kent, K. C. ; Li, L. J. Am. Soc. Mass Spectrom. 2017 , 28 , 1751 – 1764 DOI: 10.1007/s13361-017-1701-4

[Crossref], [PubMed], [CAS], Google Scholar

169

Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

Yu, Qing; Wang, Bowen; Chen, Zhengwei; Urabe, Go; Glover, Matthew S.; Shi, Xudong; Guo, Lian-Wang; Kent, K. Craig; Li, Lingjun

Journal of the American Society for Mass Spectrometry (2017), 28 (9), 1751-1764CODEN: JAMSEF; ISSN:1044-0305. (Springer)

Protein glycosylation, one of the most heterogeneous posttranslational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymically released glycans and their original peptide backbone sep., as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum, and that can be conveniently applied to high throughput glycoproteome characterization, esp. for N-glycopeptides, which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study, a redefined electron-transfer/higher-energy collision dissocn. (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding five glycosylation sites identified. Subsequent expts. tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid arteries over the course of restenosis progression to investigate the potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the anal. of intact N-glycopeptides and N-glycoproteomics. The authors also anticipate this approach can be readily applied to large-scale O-glycoproteome characterization.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFOls7bN&md5=b2eed16aa0be658929f5968ee1f9588b

170

Yu, Q. ; Canales, A. ; Glover, M. S. ; Das, R. ; Shi, X. ; Liu, Y. ; Keller, M. P. ; Attie, A. D. ; Li, L. Anal. Chem. 2017 , 89 , 9184 – 9191 DOI: 10.1021/acs.analchem.7b01926

171

Glover, M. S. ; Yu, Q. ; Chen, Z. ; Shi, X. ; Kent, K. C. ; Li, L. Int. J. Mass Spectrom. 2017 , DOI: 10.1016/j.ijms.2017.09.002

172

Parker, B. L. ; Thaysen-Andersen, M. ; Fazakerley, D. J. ; Holliday, M. ; Packer, N. H. ; James, D. E. Mol. Cell. Proteomics 2016 , 15 , 141 – 153 DOI: 10.1074/mcp.M115.054221

[Crossref], [PubMed], [CAS], Google Scholar

172

Terminal Galactosylation and Sialylation Switching on Membrane Glycoproteins upon TNF-Alpha-Induced Insulin Resistance in Adipocytes

Parker, Benjamin L.; Thaysen-Andersen, Morten; Fazakerley, Daniel J.; Holliday, Mira; Packer, Nicolle H.; James, David E.

Molecular & Cellular Proteomics (2016), 15 (1), 141-153CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Insulin resistance (IR) is a complex pathophysiol. state that arises from both environmental and genetic perturbations and leads to a variety of diseases, including type-2 diabetes (T2D). Obesity is assocd. with enhanced adipose tissue inflammation, which may play a role in disease progression. Inflammation modulates protein glycosylation in a variety of cell types, and this has been assocd. with biol. dysregulation. Here, we have examd. the effects of an inflammatory insult on protein glycosylation in adipocytes. We performed quant. N-glycome profiling of membrane proteins derived from mouse 3T3-L1 adipocytes that had been incubated with or without the proinflammatory cytokine TNF-alpha to induce IR. We identified the regulation of specific terminal N-glycan epitopes, including an increase in terminal di-galactose- and a decrease in biantennary alpha-2,3-sialoglycans. The altered N-glycosylation of TNF-alpha-treated adipocytes correlated with the regulation of specific glycosyltransferases, including the up-regulation of B4GalT5 and Ggta1 galactosyltransferases and down-regulation of ST3Gal6 sialyltransferase. Knockdown of B4GalT5 down-regulated the terminal di-galactose N-glycans, confirming the involvement of this enzyme in the TNF-alpha-regulated N-glycome. SILAC-based quant. glycoproteomics of enriched N-glycopeptides with and without deglycosylation were used to identify the protein and glycosylation sites modified with these regulated N-glycans. The combined proteome and glycoproteome workflow provided a relative quantification of changes in protein abundance vs. N-glycosylation occupancy vs. site-specific N-glycans on a proteome-wide level. This revealed the modulation of N-glycosylation on specific proteins in IR, including those previously assocd. with insulin-stimulated GLUT4 trafficking to the plasma membrane.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFOjtg%253D%253D&md5=adb6e2e974e7d541155673141972f2a7

173

Totten, S. M. ; Feasley, C. L. ; Bermudez, A. ; Pitteri, S. J. J. Proteome Res. 2017 , 16 , 1249 – 1260 DOI: 10.1021/acs.jproteome.6b00849

[ACS Full Text ], [CAS], Google Scholar

173

Parallel Comparison of N-Linked Glycopeptide Enrichment Techniques Reveals Extensive Glycoproteomic Analysis of Plasma Enabled by SAX-ERLIC

Totten, Sarah M.; Feasley, Christa L.; Bermudez, Abel; Pitteri, Sharon J.

Journal of Proteome Research (2017), 16 (3), 1249-1260CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Protein glycosylation is of increasing interest due to its important roles in protein function and aberrant expression with disease. Characterizing protein glycosylation remains anal. challenging due to its low abundance, ion suppression issues, and microheterogeneity at glycosylation sites, esp. in complex samples such as human plasma. The utility of three common N-linked glycopeptide enrichment techniques is compared using human plasma. By anal. on an LTQ-Orbitrap Elite mass spectrometer, electrostatic repulsion hydrophilic interaction liq. chromatog. using strong anion exchange solid-phase extn. (SAX-ERLIC) provided the most extensive N-linked glycopeptide enrichment when compared with multilectin affinity chromatog. (M-LAC) and Sepharose-HILIC enrichments. SAX-ERLIC enrichment yielded 191 unique glycoforms across 72 glycosylation sites from 48 glycoproteins, which is more than double that detected using other enrichment techniques. The greatest glycoform diversity was obsd. in SAX-ERLIC enrichment, with no apparent bias toward specific glycan types. SAX-ERLIC enrichments were addnl. analyzed by an Orbitrap Fusion Lumos mass spectrometer to maximize glycopeptide identifications for a more comprehensive assessment of protein glycosylation. In these expts., 829 unique glycoforms were identified across 208 glycosylation sites from 95 plasma glycoproteins, a significant improvement from the initial method comparison and one of the most extensive site-specific glycosylation anal. in immunodepleted human plasma to date. Data are available via ProteomeXchange with identifier PXD005655.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXis1agsbY%253D&md5=c19da2cdceb02dc80db191c5ce525a31

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Riley, N. M.; Hebert, A. S.; Westphall, M. S.; Coon, J. J. Thousands of Glycosites Characterized via Intact Glycopeptide Analysis using Activated Ion Electron Transfer Dissociation. In Proceedings of the 65th ASMS Conference on Mass Spectrometry and Allied Topics, Indianapolis, IN, June 4–8, 2017.

175

Bourgoin-Voillard, S. ; Leymarie, N. ; Costello, C. E. Proteomics 2014 , 14 , 1174 – 1184 DOI: 10.1002/pmic.201300433

176

Woo, C. M. ; Iavarone, A. T. ; Spiciarich, D. R. ; Palaniappan, K. K. ; Bertozzi, C. R. Nat. Methods 2015 , 12 , 561 – 567 DOI: 10.1038/nmeth.3366

[Crossref], [PubMed], [CAS], Google Scholar

176

Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis

Woo, Christina M.; Iavarone, Anthony T.; Spiciarich, David R.; Palaniappan, Krishnan K.; Bertozzi, Carolyn R.

Nature Methods (2015), 12 (6), 561-567CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

Protein glycosylation is a heterogeneous post-translational modification (PTM) that plays an essential role in biol. regulation. However, the diversity found in glycoproteins has undermined efforts to describe the intact glycoproteome via mass spectrometry (MS). We present IsoTaG, a mass-independent chem. glycoproteomics platform for characterization of intact, metabolically labeled glycopeptides at the whole-proteome scale. In IsoTaG, metabolic labeling of the glycoproteome is combined with (i) chem. enrichment and isotopic recoding of glycopeptides to select peptides for targeted glycoproteomics using directed MS and (ii) mass-independent assignment of intact glycopeptides. We structurally assigned 32 N-glycopeptides and over 500 intact and fully elaborated O-glycopeptides from 250 proteins across three human cancer cell lines and also discovered unexpected peptide sequence polymorphisms (pSPs). The IsoTaG platform is broadly applicable to the discovery of PTM sites that are amenable to chem. labeling, as well as previously unknown protein isoforms including pSPs.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXnslOnsrg%253D&md5=fc0fc8fa868caf0440d5f29069bcf08c

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Woo, C. M. ; Felix, A. ; Byrd, W. E. ; Zuegel, D. K. ; Ishihara, M. ; Azadi, P. ; Iavarone, A. T. ; Pitteri, S. J. ; Bertozzi, C. R. J. Proteome Res. 2017 , 16 , 1706 – 1718 DOI: 10.1021/acs.jproteome.6b01053

[ACS Full Text ], [CAS], Google Scholar

177

Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes

Woo, Christina M.; Felix, Alejandra; Byrd, William E.; Zuegel, Devon K.; Ishihara, Mayumi; Azadi, Parastoo; Iavarone, Anthony T.; Pitteri, Sharon J.; Bertozzi, Carolyn R.

Journal of Proteome Research (2017), 16 (4), 1706-1718CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Protein glycosylation can have an enormous variety of biol. consequences, reflecting the mol. diversity encoded in glycan structures. This same structural diversity has imposed major challenges on the development of methods to study the intact glycoproteome. The authors recently introduced a method termed isotope-targeted glycoproteomics (IsoTaG), which utilizes isotope recoding to characterize azidosugar-labeled glycopeptides bearing fully intact glycans. Here, the authors describe the broad application of the method to analyze glycoproteomes from a collection of tissue-diverse cell lines. The effort was enabled by a new high-fidelity pattern-searching and glycopeptide validation algorithm termed IsoStamp v2.0, as well as by novel stable isotope probes. Application of the IsoTaG platform to 15 cell lines metabolically labeled with Ac4GalNAz or Ac4ManNAz revealed 1375 N- and 2159 O-glycopeptides, variously modified with 74 discrete glycan structures. Glycopeptide-bound glycans obsd. by IsoTaG were found to be comparable to released N-glycans identified by permethylation anal. IsoTaG is therefore positioned to enhance structural understanding of the glycoproteome.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjtl2lsLc%253D&md5=3bf15e167bc191877f6e5f5b0965b9ae

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Woo, C. M. ; Felix, A. ; Zhang, L. ; Elias, J. E. ; Bertozzi, C. R. Anal. Bioanal. Chem. 2017 , 409 , 579 – 588 DOI: 10.1007/s00216-016-9934-9

[Crossref], [PubMed], [CAS], Google Scholar

178

Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars

Woo, Christina M.; Felix, Alejandra; Zhang, Lichao; Elias, Joshua E.; Bertozzi, Carolyn R.

Analytical and Bioanalytical Chemistry (2017), 409 (2), 579-588CODEN: ABCNBP; ISSN:1618-2642. (Springer)

Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biol. regulation. Assignment of intact glycan structures to specific protein attachment sites is a crit. step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in anal. of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1WqtbnE&md5=704e52557474d4d11c839a999a4ddb37

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Yang, Y. ; Liu, F. ; Franc, V. ; Halim, L. A. ; Schellekens, H. ; Heck, A. J. R. Nat. Commun. 2016 , 7 , 13397 DOI: 10.1038/ncomms13397

[Crossref], [PubMed], [CAS], Google Scholar

179

Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity

Yang, Yang; Liu, Fan; Franc, Vojtech; Halim, Liem Andhyk; Schellekens, Huub; Heck, Albert J. R.

Nature Communications (2016), 7 (), 13397CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)

Many biopharmaceutical products exhibit extensive structural micro-heterogeneity due to an array of co-occurring post-translational modifications. These modifications often effect the functionality of the product and therefore need to be characterized in detail. Here, we present an integrative approach, combining two advanced mass spectrometry-based methods, high-resoln. native mass spectrometry and middle-down proteomics, to analyze this micro-heterogeneity. Taking human erythropoietin and the human plasma properdin as model systems, we demonstrate that this strategy bridges the gap between peptide- and protein-based mass spectrometry platforms, providing the most complete profiling of glycoproteins. Integration of the two methods enabled the discovery of three undescribed C-glycosylation sites on properdin, and revealed in addn. unexpected heterogeneity in occupancies of C-mannosylation. Furthermore, using various sources of erythropoietin we define and demonstrate the usage of a biosimilarity score to quant. assess structural similarity, which would also be beneficial for profiling other therapeutic proteins and even plasma protein biomarkers.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVGjsL%252FI&md5=30540791a6e2417ea0e4b9cb1dae2a59

180

Franc, V. ; Yang, Y. ; Heck, A. J. R. Anal. Chem. 2017 , 89 , 3483 – 3491 DOI: 10.1021/acs.analchem.6b04527

[ACS Full Text ], [CAS], Google Scholar

180

Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation

Franc, Vojtech; Yang, Yang; Heck, Albert J. R.

Analytical Chemistry (Washington, DC, United States) (2017), 89 (6), 3483-3491CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

The human complement C9 protein (∼65 kDa) is a member of the complement pathway. It plays an essential role in the membrane attack complex (MAC), which forms a lethal pore on the cellular surface of pathogenic bacteria. Here, the authors charted in detail the structural microheterogeneity of C9 purified from human blood serum, using an integrative workflow combining high-resoln. native mass spectrometry and (glyco)peptide-centric proteomics. The proteoform profile of C9 was acquired by high-resoln. native mass spectrometry, which revealed the co-occurrence of ∼50 distinct mass spectrometry (MS) signals. Subsequent peptide-centric anal., through proteolytic digestion of C9 and liq. chromatog. (LC)-tandem mass spectrometry (MS/MS) measurements of the resulting peptide mixts., provided site-specific quant. profiles of three different types of C9 glycosylation and validation of the native MS data. The study provides a detailed specification, validation, and quantification of 15 co-occurring C9 proteoforms and the first direct exptl. evidence of O-linked glycans in the N-terminal region. Addnl., next to the two known glycosylation sites, a third novel, albeit low abundant, N-glycosylation site on C9 is identified, which surprisingly does not possess the canonical N-glycosylation sequence N-X-S/T. The data also reveal a binding of up to two Ca2+ ions to C9. Mapping all detected and validated sites of modifications on a structural model of C9, as present in the MAC, hints at their putative roles in pore formation or receptor interactions. The applied methods herein represent a powerful tool for the unbiased in-depth anal. of plasma proteins and may advance biomarker discovery, as aberrant glycosylation profiles may be indicative of the pathophysiol. state of the patients.

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Pronker, M. F. ; Lemstra, S. ; Snijder, J. ; Heck, A. J. R. ; Thies-Weesie, D. M. E. ; Pasterkamp, R. J. ; Janssen, B. J. C. Nat. Commun. 2016 , 7 , 13584 DOI: 10.1038/ncomms13584

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181

Structural basis of myelin-associated glycoprotein adhesion and signalling

Pronker Matti F; Janssen Bert J C; Lemstra Suzanne; Pasterkamp R Jeroen; Snijder Joost; Heck Albert J R; Thies-Weesie Dominique M E

Nature communications (2016), 7 (), 13584 ISSN:.

Myelin-associated glycoprotein (MAG) is a myelin-expressed cell-adhesion and bi-directional signalling molecule. MAG maintains the myelin-axon spacing by interacting with specific neuronal glycolipids (gangliosides), inhibits axon regeneration and controls myelin formation. The mechanisms underlying MAG adhesion and signalling are unresolved. We present crystal structures of the MAG full ectodomain, which reveal an extended conformation of five Ig domains and a homodimeric arrangement involving membrane-proximal domains Ig4 and Ig5. MAG-oligosaccharide complex structures and biophysical assays show how MAG engages axonal gangliosides at domain Ig1. Two post-translational modifications were identified-N-linked glycosylation at the dimerization interface and tryptophan C-mannosylation proximal to the ganglioside binding site-that appear to have regulatory functions. Structure-guided mutations and neurite outgrowth assays demonstrate MAG dimerization and carbohydrate recognition are essential for its regeneration-inhibiting properties. The combination of trans ganglioside binding and cis homodimerization explains how MAG maintains the myelin-axon spacing and provides a mechanism for MAG-mediated bi-directional signalling.

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182

Maynard, J. C. ; Burlingame, A. L. ; Medzihradszky, K. F. Mol. Cell. Proteomics 2016 , 15 , 3405 – 3411 DOI: 10.1074/mcp.M116.061549

[Crossref], [PubMed], [CAS], Google Scholar

182

Cysteine S-linked N-acetylglucosamine (S-GlcNAcylation), A New Post-translational Modification in Mammals

Maynard, Jason C.; Burlingame, Alma L.; Medzihradszky, Katalin F.

Molecular & Cellular Proteomics (2016), 15 (11), 3405-3411CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Intracellular GlcNAcylation of Ser and Thr residues is a well-known and widely investigated post-translational modification. This post-translational modification has been shown to play a significant role in cell signaling and in many regulatory processes within cells. O-GlcNAc transferase is the enzyme responsible for glycosylating cytosolic and nuclear proteins with a single GlcNAc residue on Ser and Thr side-chains. Here we report that the same enzyme may also be responsible for S-GlcNAcylation, i.e. for linking the GlcNAc unit to the peptide by modifying a cysteine side-chain. We also report that O-GlcNAcase, the enzyme responsible for removal of O-GlcNAcylation does not appear to remove the S-linked sugar. Such Cys modifications have been detected and identified in mouse and rat samples. This work has established the occurrence of 14 modification sites assigned to 11 proteins unambiguously. We have also identified S-GlcNAcylation from human Host Cell Factor 1 isolated from HEK-cells. Although these site assignments are primarily based on electron-transfer dissocn. mass spectra, we also report that S-linked GlcNAc is more stable under collisional activation than O-linked GlcNAc derivs.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhsl2gsL7N&md5=c950bc9fab9e6150dde4d5321bc63025

183

Rabbani, N. ; Ashour, A. ; Thornalley, P. J. Glycoconjugate J. 2016 , 33 , 553 – 568 DOI: 10.1007/s10719-016-9709-8

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183

Mass spectrometric determination of early and advanced glycation in biology

Rabbani, Naila; Ashour, Amal; Thornalley, Paul J.

Glycoconjugate Journal (2016), 33 (4), 553-568CODEN: GLJOEW; ISSN:0282-0080. (Springer)

Protein glycation in biol. systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quant. glycation adducts are found at mean extents of modification of 1-5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and Nε-carboxymethyl-lysine residues mainly formed by the oxidative degrdn. of fructosamine. Total glycation adducts of different types are quantified by stable isotopic diln. anal. liq. chromatog.-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metab. of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them - amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labeling with amino acids in cell culture (SILAC) high resoln. mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to anal. of glycated proteins are: (i) avoiding compromise of anal. by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric anal. for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biol., ageing and translational medicine - particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurol. disorders and cancer. Mass spectrometric anal. of glycated proteins has yet to find widespread use clin. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resoln. mass spectrometry proteomics of glycated proteins is given.

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184

Milkovska-Stamenova, S. ; Hoffmann, R. Food Chem. 2017 , 221 , 489 – 495 DOI: 10.1016/j.foodchem.2016.10.092

185

Milkovska-Stamenova, S. ; Hoffmann, R. J. Proteomics 2016 , 134 , 102 – 111 DOI: 10.1016/j.jprot.2015.12.022

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185

Hexose-derived glycation sites in processed bovine milk

Milkovska-Stamenova, Sanja; Hoffmann, Ralf

Journal of Proteomics (2016), 134 (), 102-111CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)

Milk products are consumed by many people on a daily basis, which demands sophisticated tech. processes to guarantee the microbiol. safety and to retain the nutritional value. The heating during pasteurization and ultra high temp. (UHT) treatment triggers diverse chem. reactions, such as the reaction of sugars and amino groups of proteins typically termed protein glycation. The glycation by lactose as dominant sugar in milk has been recently investigated, whereas the contribution of hexoses remains open. We identified first hexose-derived glycation sites in raw milk, colostrum, three brands of pasteurized milk, three brands of UHT milk, five brands of infant formula, and one brand of lactose-free pasteurized and UHT milk using tandem mass spectrometry and electron transfer dissocn. In total, we could identify 124 hexosylated tryptic peptides in a bottom-up proteomics approach after enriching glycated peptides by boronate affinity chromatog., which corresponded to 86 glycation sites in 17 bovine milk proteins. In quant. terms glycation increased from raw milk to pasteurized milk to UHT milk and infant formula. Lactose-free milk contained significantly higher hexosylation degrees than the corresponding regular milk product. Interestingly, the glycation degrees varied considerably among different brands with lactose-free UHT milk and infant formula showing the highest levels. The established proteomics strategy enables the identification and relative quantification of different protein glycation types in diverse milk products ranging from raw milk to milk powders. This will allow detailed in vitro studies to judge pos. or neg. aspects when consuming differently processed milk products including lactose-free milk that is obligatory for people with lactose intolerance but is increasingly consumed by the general population assuming health benefits. The established analytics will also permit studying the influence of each tech. processing step on the glycation degrees and thus offers the possibility to reduce glycation early during prodn., as obvious from the variation among different brands. Special attention should be given to the high hexose- and lactose-derived glycation levels found in infant formula, although it is still controversially discussed if protein glycation has a neg. biol. impact.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XlvVGjsw%253D%253D&md5=24260eced53774a1f5a905fea46a0bc7

186

Bern, M. ; Beniston, R. ; Mesnage, S. Anal. Bioanal. Chem. 2017 , 409 , 551 – 560 DOI: 10.1007/s00216-016-9857-5

[Crossref], [PubMed], [CAS], Google Scholar

186

Towards an automated analysis of bacterial peptidoglycan structure

Bern, Marshall; Beniston, Richard; Mesnage, Stephane

Analytical and Bioanalytical Chemistry (2017), 409 (2), 551-560CODEN: ABCNBP; ISSN:1618-2642. (Springer)

Peptidoglycan (PG) is an essential component of the bacterial cell envelope. This macromol. consists of glycan chains alternating N-acetylglucosamine and N-acetylmuramic acid, cross-linked by short peptides contg. nonstandard amino acids. Structural anal. of PG usually involves enzymic digestion of glycan strands and sepn. of disaccharide peptides by reversed-phase HPLC followed by collection of individual peaks for MALDI-TOF and/or tandem mass spectrometry. Here, we report a novel strategy using shotgun proteomics techniques for a systematic and unbiased structural anal. of PG using high-resoln. mass spectrometry and automated anal. of HCD and ETD fragmentation spectra with the Byonic software. Using the PG of the nosocomial pathogen Clostridium difficile as a proof of concept, we show that this high-throughput approach allows the identification of all PG monomers and dimers previously described, leaving only disambiguation of 3-3 and 4-3 crosslinking as a manual step. Our anal. confirms previous findings that C. difficile peptidoglycans include mainly deacetylated N-acetylglucosamine residues and 3-3 cross-links. The anal. also revealed a no. of low abundance muropeptides with peptide sequences not previously reported. [Figure not available: see fulltext.].

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187

Wu, S.-L. ; Jiang, H. ; Lu, Q. ; Dai, S. ; Hancock, W. S. ; Karger, B. L. Anal. Chem. 2009 , 81 , 112 – 122 DOI: 10.1021/ac801560k

188

Wu, S.-L. ; Jiang, H. ; Hancock, W. S. ; Karger, B. L. Anal. Chem. 2010 , 82 , 5296 – 5303 DOI: 10.1021/ac100766r

189

Massonnet, P. ; Upert, G. ; Smargiasso, N. ; Gilles, N. ; Quinton, L. ; De Pauw, E. Anal. Chem. 2015 , 87 , 5240 – 5246 DOI: 10.1021/acs.analchem.5b00245

190

Wen, D. ; Xiao, Y. ; Vecchi, M. M. ; Gong, B. J. ; Dolnikova, J. ; Pepinsky, R. B. Anal. Chem. 2017 , 89 , 4021 – 4030 DOI: 10.1021/acs.analchem.6b04600

191

Tan, L. ; Durand, K. L. ; Ma, X. ; Xia, Y. Analyst 2013 , 138 , 6759 DOI: 10.1039/c3an01333b

192

Liu, F. ; van Breukelen, B. ; Heck, A. J. R. Mol. Cell. Proteomics 2014 , 13 , 2776 – 2786 DOI: 10.1074/mcp.O114.039057

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192

Facilitating Protein Disulfide Mapping by a Combination of Pepsin Digestion, Electron Transfer Higher Energy Dissociation (EThcD), and a Dedicated Search Algorithm SlinkS

Liu, Fan; van Breukelen, Bas; Heck, Albert J. R.

Molecular & Cellular Proteomics (2014), 13 (10), 2776-2786CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Disulfide bond identification is important for a detailed understanding of protein structures, which directly affect their biol. functions. Here we describe an integrated workflow for the fast and accurate identification of authentic protein disulfide bridges. This novel workflow incorporates acidic proteolytic digestion using pepsin to eliminate undesirable disulfide reshuffling during sample prepn. and a novel search engine, SlinkS, to directly identify disulfide-bridged peptides isolated via electron transfer higher energy dissocn. (EThcD). In EThcD fragmentation of disulfide-bridged peptides, electron transfer dissocn. preferentially leads to the cleavage of the S-S bonds, generating two intense disulfide-cleaved peptides as primary fragment ions. Subsequently, higher energy collision dissocn. primarily targets unreacted and charge-reduced precursor ions, inducing peptide backbone fragmentation. SlinkS is able to provide the accurate monoisotopic precursor masses of the two disulfide-cleaved peptides and the sequence of each linked peptide by matching the remaining EThcD product ions against a linear peptide database. The workflow was validated using a protein mixt. contg. six proteins rich in natural disulfide bridges. Using this pepsin-based workflow, we were able to efficiently and confidently identify a total of 31 unique Cys-Cys bonds (out of 43 disulfide bridges present), with no disulfide reshuffling products detected. Pepsin digestion not only outperformed trypsin digestion in terms of the no. of detected authentic Cys-Cys bonds, but, more important, prevented the formation of artificially reshuffled disulfide bridges due to protein digestion under neutral pH. Our new workflow therefore provides a precise and generic approach for disulfide bridge mapping, which can be used to study protein folding, structure, and stability.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1Knsb3F&md5=250c4d8190846a90e7d6d644f2bb5e46

193

Yu, X. ; Khani, A. ; Ye, X. ; Petruzziello, F. ; Gao, H. ; Zhang, X. ; Rainer, G. Anal. Chem. 2015 , 87 , 11646 – 11651 DOI: 10.1021/ac504872z

194

Clark, D. F. ; Go, E. P. ; Desaire, H. Anal. Chem. 2013 , 85 , 1192 – 1199 DOI: 10.1021/ac303124w

195

Rombouts, I. ; Lagrain, B. ; Scherf, K. A. ; Lambrecht, M. A. ; Koehler, P. ; Delcour, J. A. Sci. Rep. 2015 , 5 , 12210 DOI: 10.1038/srep12210

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195

Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation

Rombouts, Ine; Lagrain, Bert; Scherf, Katharina A.; Lambrecht, Marlies A.; Koehler, Peter; Delcour, Jan A.

Scientific Reports (2015), 5 (), 12210CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)

Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liq. chromatog. (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissocn. (ETD) and collision-induced dissocn. (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermol. and intramol. thiol-disulfide interchange and thiol oxidn. reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chem., biochem., pharmaceutical and food processing.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlWgsr3I&md5=ec3e48c8c8b88dd3d76e2fe938984dc6

196

Trevisiol, S. ; Ayoub, D. ; Lesur, A. ; Ancheva, L. ; Gallien, S. ; Domon, B. Proteomics 2016 , 16 , 715 – 728 DOI: 10.1002/pmic.201500379

197

Tsiatsiani, L. ; Heck, A. J. R. FEBS J. 2015 , 282 , 2612 – 2626 DOI: 10.1111/febs.13287

[Crossref], [PubMed], [CAS], Google Scholar

197

Proteomics beyond trypsin

Tsiatsiani, Liana; Heck, Albert J. R.

FEBS Journal (2015), 282 (14), 2612-2626CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)

A review. Peptide-centered shotgun anal. of proteins has been the core technol. in mass spectrometry based proteomics and has enabled numerous biol. discoveries, such as the large-scale charting of protein-protein interaction networks, the quant. anal. of protein post-translational modifications and even the first drafts of the human proteome. The conversion of proteins into peptides in these so-called bottom-up approaches is nearly uniquely done by using trypsin as a proteolytic reagent. Here, the authors argue that the authors' view of the proteome still remains incomplete and this is partially due to the nearly exclusive use of trypsin. Newly emerging alternative proteases and/or multi-protease protein digestion aim to increase proteome sequence coverage and improve the identification of post-translational modifications, through the anal. of complementary and often longer peptides, introducing an approach termed middle-down proteomics. Of pivotal importance for this purpose is the identification of proteases beneficial for use in proteomics. Here, the authors describe some of the shortcomings of the nearly exclusive use of trypsin in proteomics and review the properties of other proteomics-appropriate proteases. The authors describe favorable protease traits with an emphasis on middle-down proteomics and suggest potential sources for the discovery of new proteases. The authors also highlight a few examples wherein the use of other proteases than trypsin enabled the generation of more comprehensive data sets leading to previously unexplored knowledge of the proteome.

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198

Swaney, D. L. ; Wenger, C. D. ; Coon, J. J. J. Proteome Res. 2010 , 9 , 1323 – 1329 DOI: 10.1021/pr900863u

[ACS Full Text ], [CAS], Google Scholar

198

Value of Using Multiple Proteases for Large-Scale Mass Spectrometry-Based Proteomics

Swaney, Danielle L.; Wenger, Craig D.; Coon, Joshua J.

Journal of Proteome Research (2010), 9 (3), 1323-1329CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Large-scale protein sequencing methods rely on enzymic digestion of complex protein mixts. to generate a collection of peptides for mass spectrometric anal. Here the authors examine the use of multiple proteases (trypsin, LysC, ArgC, AspN, and GluC) to improve both protein identification and characterization in the model organism Saccharomyces cerevisiae. Using a data-dependent, decision tree-based algorithm to tailor MS2 fragmentation method to peptide precursor, the authors identified 92,095 unique peptides (609,665 total) mapping to 3908 proteins at a 1% false discovery rate (FDR). These results were a significant improvement upon data from a single protease digest (trypsin) - 27,822 unique peptides corresponding to 3313 proteins. The addnl. 595 protein identifications were mainly from those at low abundances (i.e., <1000 copies/cell); sequence coverage for these proteins was likewise improved nearly 3-fold. The authors demonstrate that large portions of the proteome are simply inaccessible following digestion with a single protease and that multiple proteases, rather than tech. replicates, provide a direct route to increase both protein identifications and proteome sequence coverage.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXht12hs7o%253D&md5=cc2acd199ffedfe39ce181218625f909

199

Nardiello, D. ; Palermo, C. ; Natale, A. ; Quinto, M. ; Centonze, D. Anal. Chim. Acta 2015 , 854 , 106 – 117 DOI: 10.1016/j.aca.2014.10.053

[Crossref], [PubMed], [CAS], Google Scholar

199

Strategies in protein sequencing and characterization: Multi-enzyme digestion coupled with alternate CID/ETD tandem mass spectrometry

Nardiello, Donatella; Palermo, Carmen; Natale, Anna; Quinto, Maurizio; Centonze, Diego

Analytica Chimica Acta (2015), 854 (), 106-117CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)

A strategy based on a simultaneous multi-enzyme digestion coupled with electron transfer dissocn. (ETD) and collision-induced dissocn. (CID) was developed for protein sequencing and characterization, as a valid alternative platform in ion-trap based proteomics. The effect of different proteolytic procedures using chymotrypsin, trypsin, a combination of both, and Lys-C, was carefully evaluated in terms of no. of identified peptides, protein coverage, and score distribution. A systematic comparison between CID and ETD is shown for the anal. of peptides originating from the in-soln. digestion of std. caseins. The best results were achieved with a trypsin/chymotrypsin mix combined with CID and ETD operating in alternating mode. A post-database search validation of MS/MS dataset was performed, then, the matched peptides were cross checked by the evaluation of ion scores, rank, no. of exptl. product ions, and their relative abundances in the MS/MS spectrum. By integrated CID/ETD expts., high quality-spectra have been obtained, thus allowing a confirmation of spectral information and an increase of accuracy in peptide sequence assignments. Overlapping peptides, produced throughout the proteins, reduce the ambiguity in mapping modifications between natural variants and animal species, and allow the characterization of post translational modifications. The advantages of using the enzymic mix trypsin/chymotrypsin were confirmed by the nanoLC and CID/ETD tandem mass spectrometry of goat milk proteins, previously sepd. by two-dimensional gel electrophoresis.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVGktLnM&md5=8455adffca9a87d3b897da6d06113b26

200

Somasundaram, P. ; Koudelka, T. ; Linke, D. ; Tholey, A. J. Proteome Res. 2016 , 15 , 1369 – 1378 DOI: 10.1021/acs.jproteome.6b00146

[ACS Full Text ], [CAS], Google Scholar

200

C-Terminal Charge-Reversal Derivatization and Parallel Use of Multiple Proteases Facilitates Identification of Protein C-Termini by C-Terminomics

Somasundaram, Prasath; Koudelka, Tomas; Linke, Dennis; Tholey, Andreas

Journal of Proteome Research (2016), 15 (4), 1369-1378CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

The identification of protein C-termini in complex proteomes is challenging due to the poor ionization efficiency of the carboxyl group. Amidating the neg. charged C-termini with ethanolamine (EA) has been suggested to improve the detection of C-terminal peptides and allows for a directed depletion of internal peptides after proteolysis using carboxyl reactive polymers. In the present study, the derivatization with N,N-dimethylethylenediamine (DMEDA) and (4-aminobutyl)guanidine (AG) leading to a pos. charged C-terminus was investigated. C-terminal charge-reversed peptides showed improved coverage of b- and y-ion series in the MS/MS spectra compared to their noncharged counterparts. DMEDA-derivatized peptides resulted in many peptides with charge states of 3+, which benefited from ETD fragmentation. This makes the charge-reversal strategy particularly useful for the anal. of protein C-termini, which may also be post-translationally modified. The labeling strategy and the indirect enrichment of C-termini worked with similar efficiency for both DMEDA and EA, and their applicability was demonstrated on an E. coli proteome. Utilizing two proteases and different MS/MS activation mechanisms allowed for the identification of >400 C-termini, encompassing both canonical and truncated C-termini.

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201

van der Post, S. ; Thomsson, K. A. ; Hansson, G. C. J. Proteome Res. 2014 , 13 , 6013 – 6023 DOI: 10.1021/pr500874f

[ACS Full Text ], [CAS], Google Scholar

201

Multiple Enzyme Approach for the Characterization of Glycan Modifications on the C-Terminus of the Intestinal MUC2Mucin

van der Post, Sjoerd; Thomsson, Kristina A.; Hansson, Gunnar C.

Journal of Proteome Research (2014), 13 (12), 6013-6023CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein's central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein contg. the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 N-glycan consensus sites were identified as glycosylated. Fifty-six N-glycopeptides covering 10 N-glycan sites, and 14 O-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the d. and complexity of glycosylation of complex glycoproteins such as mucins.

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202

Guthals, A. ; Clauser, K. R. ; Frank, A. M. ; Bandeira, N. J. Proteome Res. 2013 , 12 , 2846 – 2857 DOI: 10.1021/pr400173d

[ACS Full Text ], [CAS], Google Scholar

202

Sequencing-Grade De novo Analysis of MS/MS Triplets (CID/HCD/ETD) From Overlapping Peptides

Guthals, Adrian; Clauser, Karl R.; Frank, Ari M.; Bandeira, Nuno

Journal of Proteome Research (2013), 12 (6), 2846-2857CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Full-length de novo sequencing of unknown proteins remains a challenging open problem. Traditional methods that sequence spectra individually are limited by short peptide length, incomplete peptide fragmentation, and ambiguous de novo interpretations. The authors address these issues by detg. consensus sequences for assembled tandem mass (MS/MS) spectra from overlapping peptides (e.g., by using multiple enzymic digests). The authors have combined electron-transfer dissocn. (ETD) with collision-induced dissocn. (CID) and higher-energy collision-induced dissocn. (HCD) fragmentation methods to boost interpretation of long, highly charged peptides and take advantage of corroborating b/y/c/z ions in CID/HCD/ETD. Using these strategies, triplet CID/HCD/ETD MS/MS spectra from overlapping peptides yield de novo sequences of av. length 70 AA and as long as 200 AA at up to 99% sequencing accuracy.

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203

Tsiatsiani, L. ; Giansanti, P. ; Scheltema, R. A. ; van den Toorn, H. ; Overall, C. M. ; Altelaar, A. F. M. ; Heck, A. J. R. J. Proteome Res. 2017 , 16 , 852 – 861 DOI: 10.1021/acs.jproteome.6b00825

[ACS Full Text ], [CAS], Google Scholar

203

Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X)n and (X)nK/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics

Tsiatsiani, Liana; Giansanti, Piero; Scheltema, Richard A.; van den Toorn, Henk; Overall, Christopher M.; Altelaar, A. F. Maarten; Heck, Albert J. R.

Journal of Proteome Research (2017), 16 (2), 852-861CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissocn. (CID) or higher-energy collisional dissocn. (HCD) because the fragmentation rules are well-understood. Here, the authors study LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, the authors compare side-by-side the chromatog. sepn. properties, gas-phase fragmentation characteristics, and (phospho)proteome sequence coverage of tryptic (i.e., (X)nK/R) and LysargiNase (i.e., K/R(X)n) peptides using primarily electron-transfer dissocn. (ETD) and, for comparison, HCD. Tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions (ETD) and b-ions (HCD)) are formed, whereas for trypsin, C-terminal fragment ions dominate (z-ions (ETD) and y-ions (HCD)) in a homologous mixt. of complementary ions. Esp. during ETD, LysargiNase peptides fragment into low-complexity but information-rich sequence ladders. Trypsin and LysargiNase chart distinct parts of the proteome, and therefore, the combined use of these enzymes will benefit a more in-depth and reliable anal. of (phospho)proteomes.

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204

Ebhardt, H. A. ; Nan, J. ; Chaulk, S. G. ; Fahlman, R. P. ; Aebersold, R. Rapid Commun. Mass Spectrom. 2014 , 28 , 2735 – 2743 DOI: 10.1002/rcm.7069

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204

Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage

Ebhardt, H. Alexander; Nan, Jie; Chaulk, Steven G.; Fahlman, Richard P.; Aebersold, Ruedi

Rapid Communications in Mass Spectrometry (2014), 28 (24), 2735-2743CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)

RATIONALE : Tandem mass (MS/MS) spectra generated by collision-induced dissocn. (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues. METHODS : Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNAArg onto the N-terminus of the substrate peptide. This enzymic reaction is carried out under physiol. conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini. RESULTS : We demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissocn. (ETD) despite having an internal proline residue. CONCLUSIONS : We introduce a rapid enzymic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern. .

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVyrtL3O&md5=26609311dc25cd648f7d1d5905b4240d

205

Sanders, J. D. ; Greer, S. M. ; Brodbelt, J. S. Anal. Chem. 2017 , 89 , 11772 – 11778 DOI: 10.1021/acs.analchem.7b03396

206

Wu, C. ; Tran, J. C. ; Zamdborg, L. ; Durbin, K. R. ; Li, M. ; Ahlf, D. R. ; Early, B. P. ; Thomas, P. M. ; Sweedler, J. V. ; Kelleher, N. L. Nat. Methods 2012 , 9 , 822 – 824 DOI: 10.1038/nmeth.2074

[Crossref], [PubMed], [CAS], Google Scholar

206

A protease for 'middle-down' proteomics

Wu, Cong; Tran, John C.; Zamdborg, Leonid; Durbin, Kenneth R.; Li, Mingxi; Ahlf, Dorothy R.; Early, Bryan P.; Thomas, Paul M.; Sweedler, Jonathan V.; Kelleher, Neil L.

Nature Methods (2012), 9 (8), 822-824CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

We developed a method for restricted enzymic proteolysis using the outer membrane protease T (OmpT) to produce large peptides (>6.3 kDa on av.) for mass spectrometry-based proteomics. Using this approach to analyze prefractionated high-mass HeLa proteins, we identified 3,697 unique peptides from 1,038 proteins. We demonstrated the ability of large OmpT peptides to differentiate closely related protein isoforms and to enable the detection of many post-translational modifications.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xos12hs7k%253D&md5=12188a820e7b62bd4059654c2432e850

207

Cristobal, A. ; Marino, F. ; Post, H. ; van den Toorn, H. W. P. ; Mohammed, S. ; Heck, A. J. R. Anal. Chem. 2017 , 89 , 3318 – 3325 DOI: 10.1021/acs.analchem.6b03756

[ACS Full Text ], [CAS], Google Scholar

207

Toward an Optimized Workflow for Middle-Down Proteomics

Cristobal, Alba; Marino, Fabio; Post, Harm; van den Toorn, Henk W. P.; Mohammed, Shabaz; Heck, Albert J. R.

Analytical Chemistry (Washington, DC, United States) (2017), 89 (6), 3318-3325CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, either targeting relatively small peptides (i.e., 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < Mw < 30 kDa), resp. termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the anal. of middle-range peptides (i.e., 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, the authors hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the anal. of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates the authors explored the use of the proteases Asp-N and Glu-C, and a non-enzymic acid induced cleavage. To increase the depth of the proteome, an SCX sepn., carefully tuned to improve the sepn. of longer peptides, combined with RP-LC using columns packed with material possessing a larger pore size were used. Finally, after evaluating the combination of potentially beneficial MS settings, the authors also assessed the peptide fragmentation techniques HCD, ETD and EThcD for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, amongst other things, the enhanced anal. of (co-occurring) post-translational modifications.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjtlCnsro%253D&md5=b1ab30bcb0586bf21a7dee1eaa5814a7

208

Dallas, D. C. ; Guerrero, A. ; Parker, E. A. ; Robinson, R. C. ; Gan, J. ; German, J. B. ; Barile, D. ; Lebrilla, C. B. Proteomics 2015 , 15 , 1026 – 1038 DOI: 10.1002/pmic.201400310

209

Sasaki, K. ; Osaki, T. ; Minamino, N. Mol. Cell. Proteomics 2013 , 12 , 700 – 709 DOI: 10.1074/mcp.M112.017400

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209

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

Sasaki, Kazuki; Osaki, Tsukasa; Minamino, Naoto

Molecular & Cellular Proteomics (2013), 12 (3), 700-709CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Mass spectrometry-based unbiased anal. of the full complement of secretory peptides is expected to facilitate the identification of unknown biol. active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissocn. (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissocn. (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), resp., with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjtlOit7c%253D&md5=50bb24c86921075ef28ab04b79378d87

210

Li, W. ; Petruzziello, F. ; Zhao, N. ; Zhao, H. ; Ye, X. ; Zhang, X. ; Rainer, G. Proteomics 2017 , 17 , 1600419 DOI: 10.1002/pmic.201600419

211

Frese, C. K. ; Boender, A. J. ; Mohammed, S. ; Heck, A. J. R. ; Adan, R. A. H. ; Altelaar, A. F. M. Anal. Chem. 2013 , 85 , 4594 – 4604 DOI: 10.1021/ac400232y

212

Hart, S. R. ; Kenny, L. C. ; Myers, J. E. ; Baker, P. N. Int. J. Mass Spectrom. 2015 , 391 , 41 – 46 DOI: 10.1016/j.ijms.2015.08.025

213

Gucinski, A. C. ; Boyne, M. T. Rapid Commun. Mass Spectrom. 2014 , 28 , 1757 – 1763 DOI: 10.1002/rcm.6957

[Crossref], [PubMed], [CAS], Google Scholar

213

Identification of site-specific heterogeneity in peptide drugs using intact mass spectrometry with electron transfer dissociation

Gucinski, Ashley C.; Boyne, Michael T., II

Rapid Communications in Mass Spectrometry (2014), 28 (15), 1757-1763CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)

RATIONALE : Protamine sulfate is a peptide drug product consisting of multiple basic peptides. As traditional high-performance liq. chromatog. (HPLC) sepn. methods may not resolve these peptides, as well as any possible peptide-related impurities, a method utilizing top-down mass spectrometry was developed for the characterization of complex peptide drug products, including any low-level impurities, which is described in this study. METHODS : Herring protamine sulfate was used as a model system to demonstrate the applicability of the method. Direct infusion mass spectrometry and tandem mass spectrometry (MS/MS) on a high-resoln., mass accurate instrument with electron transfer dissocn. (ETD) were used to identify all the species present in the herring protamine sulfate sample. Identifications were made based on mass accuracy anal. as well as MS/MS fragmentation patterns. RESULTS : Complete sequence coverage of the three abundant herring protamine peptides was obtained using the top-down ETD-MS/MS method, which also identified a discrepancy with the published herring protamine peptide sequences. Addnl., three low-abundance related peptide species were also identified and fully characterized. These three peptides had not previously been reported as herring protamine peptides, but could be related to the published sequences through amino acid addns. and/or substitutions. CONCLUSIONS : A method for the characterization of protamine, a complex peptide drug product, was developed that can be extended to other complex peptide or protein drug products. The selectivity and sensitivity of this method improves a regulator's ability to identify peptide impurities not previously obsd. using the established methods and presents an opportunity to better understand the compn. of complex peptide drug products. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtVOjsbfM&md5=e760ec28346bbd406f861e38146ebbf2

214

Juba, M. L. ; Russo, P. S. ; Devine, M. ; Barksdale, S. ; Rodriguez, C. ; Vliet, K. A. ; Schnur, J. M. ; van Hoek, M. L. ; Bishop, B. M. J. Proteome Res. 2015 , 14 , 4282 – 4295 DOI: 10.1021/acs.jproteome.5b00447

[ACS Full Text ], [CAS], Google Scholar

214

Large Scale Discovery and De Novo-Assisted Sequencing of Cationic Antimicrobial Peptides (CAMPs) by Microparticle Capture and Electron-Transfer Dissociation (ETD) Mass Spectrometry

Juba, Melanie L.; Russo, Paul S.; Devine, Megan; Barksdale, Stephanie; Rodriguez, Carlos; Vliet, Kent A.; Schnur, Joel M.; vanHoek, Monique L.; Bishop, Barney M.

Journal of Proteome Research (2015), 14 (10), 4282-4295CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

The identification and sequencing of novel cationic antimicrobial peptides (CAMPs) have proven challenging due to the limitations assocd. with traditional proteomics methods and difficulties sequencing peptides present in complex biomol. mixts. The authors present here a process for large-scale identification and de novo-assisted sequencing of newly discovered CAMPs using microparticle capture followed by tandem mass spectrometry equipped with electron-transfer dissocn. (ETD). This process was initially evaluated and verified using known CAMPs with varying physicochem. properties. The effective parameters were then applied in the anal. of a complex mixt. of peptides harvested from American alligator plasma using custom-made (Bioprospector) functionalized hydrogel particles. Here, the authors report the successful sequencing process for CAMPs that led to the identification of 340 unique peptides and the discovery of five novel CAMPs from American alligator plasma.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVart7rK&md5=d43088ccc1001810c9338bb1604dc226

215

Bishop, B. M. ; Juba, M. L. ; Russo, P. S. ; Devine, M. ; Barksdale, S. M. ; Scott, S. ; Settlage, R. ; Michalak, P. ; Gupta, K. ; Vliet, K. ; Schnur, J. M. ; van Hoek, M. L. J. Proteome Res. 2017 , 16 , 1470 – 1482 DOI: 10.1021/acs.jproteome.6b00857

[ACS Full Text ], [CAS], Google Scholar

215

Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry

Bishop, Barney M.; Juba, Melanie L.; Russo, Paul S.; Devine, Megan; Barksdale, Stephanie M.; Scott, Shaylyn; Settlage, Robert; Michalak, Pawel; Gupta, Kajal; Vliet, Kent; Schnur, Joel M.; van Hoek, Monique L.

Journal of Proteome Research (2017), 16 (4), 1470-1482CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Komodo dragons are the largest living lizards and are the apex predators in their environs. They endure numerous strains of pathogenic bacteria in their saliva and recover from wounds inflicted by other dragons, reflecting the inherent robustness of their innate immune defense. We have employed a custom bioprospecting approach combining partial de novo peptide sequencing with transcriptome assembly to identify cationic antimicrobial peptides from Komodo dragon plasma. Through these analyses, we identified 48 novel potential cationic antimicrobial peptides. All but one of the identified peptides were derived from histone proteins. The antimicrobial effectiveness of eight of these peptides was evaluated against Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 25923), with seven peptides exhibiting antimicrobial activity against both microbes and one only showing significant potency against P. aeruginosa. This study demonstrates the power and promise of our bioprospecting approach to cationic antimicrobial peptide discovery, and it reveals the presence of a plethora of novel histone-derived antimicrobial peptides in the plasma of the Komodo dragon. These findings may have broader implications regarding the role that intact histones and histone-derived peptides play in defending the host from infection. Data are available via ProteomeXChange with identifier PXD005043.

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216

Trevisan-Silva, D. ; Bednaski, A. V. ; Fischer, J. S. G. ; Veiga, S. S. ; Bandeira, N. ; Guthals, A. ; Marchini, F. K. ; Leprevost, F. V. ; Barbosa, V. C. ; Senff-Ribeiro, A. ; Carvalho, P. C. Sci. Data 2017 , 4 , 170090 DOI: 10.1038/sdata.2017.90

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216

A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the Loxosceles intermedia venom

Trevisan-Silva, Dilza; Bednaski, Aline V.; Fischer, Juliana S. G.; Veiga, Silvio S.; Bandeira, Nuno; Guthals, Adrian; Marchini, Fabricio K.; Leprevost, Felipe V.; Barbosa, Valmir C.; Senff-Ribeiro, Andrea; Carvalho, Paulo C.

Scientific Data (2017), 4 (), 170090CODEN: SDCABS; ISSN:2052-4463. (Nature Publishing Group)

Venoms are a rich source for the discovery of mols. with biotechnol. applications, but their anal. is challenging even for state-of-the-art proteomics. Here we report on a large-scale proteomic assessment of the venom of Loxosceles intermedia, the so-called brown spider. Venom was extd. from 200 spiders and fractioned into two aliquots relative to a 10 kDa cutoff mass. Each of these was further fractioned and digested with trypsin (4 h), trypsin (18 h), pepsin (18 h), and chymotrypsin (18 h), then analyzed by MudPIT on an LTQ-Orbitrap XL ETD mass spectrometer fragmenting precursors by CID, HCD, and ETD. Aliquots of undigested samples were also analyzed. Our exptl. design allowed us to apply spectral networks, thus enabling us to obtain meta-contig assemblies, and consequently de novo sequencing of practically complete proteins, culminating in a deep proteome assessment of the venom. Data are available via ProteomeXchange, with identifier PXD005523.

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217

Hunt, D. F. ; Henderson, R. A. ; Shabanowitz, J. ; Sakaguchi, K. ; Michel, H. ; Sevilir, N. ; Cox, A. L. ; Appella, E. ; Engelhard, V. H. Science 1992 , 255 , 1261 – 1263 DOI: 10.1126/science.1546328

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217

Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry

Hunt, Donald F.; Henderson, Robert A.; Shabanowitz, Jeffrey; Sakaguchi, Kazuyasu; Michel, Hanspeter; Sevilir, Noelle; Cox, Andrea L.; Appella, Ettore; Engelhard, Victor H.

Science (Washington, DC, United States) (1992), 255 (5049), 1261-3CODEN: SCIEAS; ISSN:0036-8075.

Antigens recognized by T cells are expressed as peptides bound to major histocompatibility complex (MHC) mols. Microcapillary high-performance liq. chromatog.-electrospray ionization-tandem mass spectrometry was used to fractionate and sequence subpicomolar amts. of peptides isolated from the MHC mol. HLA-A2.1. Of 200 different species quantitated, eight were sequenced and four were found in cellular proteins. All were nine residues long and shared a distinct structural motif. The sensitivity and speed of this approach should enhance the anal. of peptides from small quantities of virally infected and transformed cells as well as those assocd. with autoimmune disease states.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XhvFahsr0%253D&md5=64e4938532fd34a5edd5ecda6b7598e3

218

Abelin, J. G. ; Trantham, P. D. ; Penny, S. A. ; Patterson, A. M. ; Ward, S. T. ; Hildebrand, W. H. ; Cobbold, M. ; Bai, D. L. ; Shabanowitz, J. ; Hunt, D. F. Nat. Protoc. 2015 , 10 , 1308 – 1318 DOI: 10.1038/nprot.2015.086

219

Cobbold, M. ; De La Peña, H. ; Norris, A. ; Polefrone, J. M. ; Qian, J. ; English, A. M. ; Cummings, K. L. ; Penny, S. ; Turner, J. E. ; Cottine, J. ; Abelin, J. G. ; Malaker, S. A. ; Zarling, A. L. ; Huang, H.-W. ; Goodyear, O. ; Freeman, S. D. ; Shabanowitz, J. ; Pratt, G. ; Craddock, C. ; Williams, M. E. ; Hunt, D. F. ; Engelhard, V. H. Sci. Transl. Med. 2013 , 5 , 203ra125 DOI: 10.1126/scitranslmed.3006061

220

Mommen, G. P. M. ; Marino, F. ; Meiring, H. D. ; Poelen, M. C. M. ; van Gaans-van den Brink, J. A. M. ; Mohammed, S. ; Heck, A. J. R. ; van Els, C. A. C. M. Mol. Cell. Proteomics 2016 , 15 , 1412 – 1423 DOI: 10.1074/mcp.M115.055780

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220

Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity

Mommen, Geert P. M.; Marino, Fabio; Meiring, Hugo D.; Poelen, Martien C. M.; van Gaans-van den Brink, Jacqueline A. M.; Mohammed, Shabaz; Heck, Albert J. R.; van Els, Cecile A. C. M.

Molecular & Cellular Proteomics (2016), 15 (4), 1412-1423CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Comprehensive anal. of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4+ T cell mediated immunity and tolerance. Here, we implemented important improvements in the anal. of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a tech. viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of phys. chem. properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity assocd. to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biol. events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunol. studies in characterizing the full breadth of potential CD4+ T cell epitopes relevant in health and disease.

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221

Malaker, S. A. ; Penny, S. A. ; Steadman, L. G. ; Myers, P. T. ; Loke, J. C. ; Raghavan, M. ; Bai, D. L. ; Shabanowitz, J. ; Hunt, D. F. ; Cobbold, M. Cancer Immunol. Res. 2017 , 5 , 376 – 384 DOI: 10.1158/2326-6066.CIR-16-0280

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221

Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia

Malaker, Stacy A.; Penny, Sarah A.; Steadman, Lora G.; Myers, Paisley T.; Loke, Justin C.; Raghavan, Manoj; Bai, Dina L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Cobbold, Mark

Cancer Immunology Research (2017), 5 (5), 376-384CODEN: CIRACV; ISSN:2326-6066. (American Association for Cancer Research)

Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I-assocd. peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three exptl. approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be assocd. with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equiv. unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376-84. ©2017 AACR.

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222

Marino, F. ; Bern, M. ; Mommen, G. P. M. ; Leney, A. C. ; van Gaans-van den Brink, J. A. M. ; Bonvin, A. M. J. J. ; Becker, C. ; van Els, C. A. C. M. ; Heck, A. J. R. J. Am. Chem. Soc. 2015 , 137 , 10922 – 10925 DOI: 10.1021/jacs.5b06586

223

Marino, F. ; Mommen, G. P. M. ; Jeko, A. ; Meiring, H. D. ; van Gaans-van den Brink, J. A. M. ; Scheltema, R. A. ; van Els, C. A. C. M. ; Heck, A. J. R. J. Proteome Res. 2017 , 16 , 34 – 44 DOI: 10.1021/acs.jproteome.6b00528

[ACS Full Text ], [CAS], Google Scholar

223

Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07

Marino, Fabio; Mommen, Geert P. M.; Jeko, Anita; Meiring, Hugo D.; van Gaans-van den Brink, Jacqueline A. M.; Scheltema, Richard A.; van Els, Cecile A. C. M.; Heck, Albert J. R.

Journal of Proteome Research (2017), 16 (1), 34-44CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Alterations in protein post-translational modification (PTMs) are recognized hallmarks of diseases. These modifications potentially provide a unique source of disease-related Human Leukocyte Antigen (HLA) class I-presented peptides that can elicit specific immune responses. While phosphorylated HLA peptides have already received attention, arginine methylated HLA class I peptide presentation has not been characterized in detail. In a human B-cell line the authors detected 149 HLA class I peptides harboring mono- and/or di-methylated arginine residues by mass spectrometry. A striking preference was obsd. in presentation of arginine (di)methylated peptides for HLA-B*07 mols., likely because the binding motifs of this allele resemble consensus sequences recognized by arginine methyl-transferases. Moreover, HLA-B*07-bound peptides preferentially harbored di-methylated groups at the P3 position, thus consecutively to the proline anchor residue. Such a proline-arginine sequence has been assocd. with the arginine methyl-transferases CARM1 and PRMT5. Making use of the specific neutral losses in fragmentation spectra the authors found most of the peptides to be asym. di-methylated, most likely by CARM1. These data expand the authors' knowledge of the processing and presentation of arginine (di)methylated HLA class I peptides, and demonstrate that these types of modified peptides can be presented for recognition by T-cells. HLA class I peptides with mono- and di-methylated arginine residues may therefore offer a novel target for immunotherapy.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht12murfM&md5=7d6dd0de724666b47137ea2eebdef688

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Liepe, J. ; Marino, F. ; Sidney, J. ; Jeko, A. ; Bunting, D. E. ; Sette, A. ; Kloetzel, P. M. ; Stumpf, M. P. H. ; Heck, A. J. R. ; Mishto, M. Science 2016 , 354 , 354 – 358 DOI: 10.1126/science.aaf4384

225

Pavlos, R. ; McKinnon, E. J. ; Ostrov, D. A. ; Peters, B. ; Buus, S. ; Koelle, D. ; Chopra, A. ; Schutte, R. ; Rive, C. ; Redwood, A. ; Restrepo, S. ; Bracey, A. ; Kaever, T. ; Myers, P. ; Speers, E. ; Malaker, S. A. ; Shabanowitz, J. ; Jing, Y. ; Gaudieri, S. ; Hunt, D. F. ; Carrington, M. ; Haas, D. W. ; Mallal, S. ; Phillips, E. J. Sci. Rep. 2017 , 7 , 8653 DOI: 10.1038/s41598-017-08876-0

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225

Shared peptide binding of HLA Class I and II alleles associate with cutaneous nevirapine hypersensitivity and identify novel risk alleles

Pavlos Rebecca; McKinnon Elizabeth J; Chopra Abha; Rive Craig; Redwood Alec; Gaudieri Silvana; Mallal Simon; Phillips Elizabeth J; Ostrov David A; Schutte Ryan; Restrepo Susana; Bracey Austin; Peters Bjoern; Kaever Thomas; Buus Soren; Koelle David; Koelle David; Koelle David; Koelle David; Koelle David; Myers Paisley; Speers Ellen; Malaker Stacy A; Shabanowitz Jeffrey; Hunt Donald F; Jing Yuan; Gaudieri Silvana; Gaudieri Silvana; Haas David W; Mallal Simon; Phillips Elizabeth J; Carrington Mary; Carrington Mary; Carrington Mary; Haas David W

Scientific reports (2017), 7 (1), 8653 ISSN:.

Genes of the human leukocyte antigen (HLA) system encode cell-surface proteins involved in regulation of immune responses, and the way drugs interact with the HLA peptide binding groove is important in the immunopathogenesis of T-cell mediated drug hypersensitivity syndromes. Nevirapine (NVP), is an HIV-1 antiretroviral with treatment-limiting hypersensitivity reactions (HSRs) associated with multiple class I and II HLA alleles. Here we utilize a novel analytical approach to explore these multi-allelic associations by systematically examining HLA molecules for similarities in peptide binding specificities and binding pocket structure. We demonstrate that primary predisposition to cutaneous NVP HSR, seen across ancestral groups, can be attributed to a cluster of HLA-C alleles sharing a common binding groove F pocket with HLA-C*04:01. An independent association with a group of class II alleles which share the HLA-DRB1-P4 pocket is also observed. In contrast, NVP HSR protection is afforded by a cluster of HLA-B alleles defined by a characteristic peptide binding groove B pocket. The results suggest drug-specific interactions within the antigen binding cleft can be shared across HLA molecules with similar binding pockets. We thereby provide an explanation for multiple HLA associations with cutaneous NVP HSR and advance insight into its pathogenic mechanisms.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cfpsVyitA%253D%253D&md5=e5e83609934c376840556e624e467163

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Malaker, S. A. ; Ferracane, M. J. ; Depontieu, F. R. ; Zarling, A. L. ; Shabanowitz, J. ; Bai, D. L. ; Topalian, S. L. ; Engelhard, V. H. ; Hunt, D. F. J. Proteome Res. 2017 , 16 , 228 – 237 DOI: 10.1021/acs.jproteome.6b00496

[ACS Full Text ], [CAS], Google Scholar

226

Identification and Characterization of Complex Glycosylated Peptides Presented by the MHC Class II Processing Pathway in Melanoma

Malaker, Stacy A.; Ferracane, Michael J.; Depontieu, Florence R.; Zarling, Angela L.; Shabanowitz, Jeffrey; Bai, Dina L.; Topalian, Suzanne L.; Engelhard, Victor H.; Hunt, Donald F.

Journal of Proteome Research (2017), 16 (1), 228-237CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

The MHC class II (MHCII) processing pathway presents peptides derived from exogenous or membrane-bound proteins to CD4+ T cells. Several studies have shown that glycopeptides are necessary to modulate CD4+ T cell recognition, though glycopeptide structures in these cases are generally unknown. Here, we present a total of 93 glycopeptides from three melanoma cell lines and one matched EBV-transformed line with most found only in the melanoma cell lines. The glycosylation we detected was diverse and comprised 17 different glycoforms. We then used mol. modeling to demonstrate that complex glycopeptides are capable of binding the MHC and may interact with complementarity detg. regions. Finally, we present the first evidence of disulfide-bonded peptides presented by MHCII. This is the first large scale study to sequence glyco- and disulfide bonded MHCII peptides from the surface of cancer cells and could represent a novel avenue of tumor activation and/or immunoevasion.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlOqurnE&md5=4c46e02e551233f6152cc53abfcfad06

227

Moradian, A. ; Kalli, A. ; Sweredoski, M. J. ; Hess, S. Proteomics 2014 , 14 , 489 – 497 DOI: 10.1002/pmic.201300256

228

Önder, Ö. ; Sidoli, S. ; Carroll, M. ; Garcia, B. A. Expert Rev. Proteomics 2015 , 12 , 499 – 517 DOI: 10.1586/14789450.2015.1084231

[Crossref], [PubMed], [CAS], Google Scholar

228

Progress in epigenetic histone modification analysis by mass spectrometry for clinical investigations

Onder, Ozlem; Sidoli, Simone; Carroll, Martin; Garcia, Benjamin A.

Expert Review of Proteomics (2015), 12 (5), 499-517CODEN: ERPXA3; ISSN:1478-9450. (Taylor & Francis Ltd.)

A review. Chromatin biol. and epigenetics are scientific fields that are rapid expanding due to their fundamental role in understanding cell development, heritable characters and progression of diseases. Histone post-translational modifications (PTMs) are major regulators of the epigenetic machinery due to their ability to modulate gene expression, DNA repair and chromosome condensation. Large-scale strategies based on mass spectrometry have been impressively improved in the last decade, so that global changes of histone PTM abundances are quantifiable with nearly routine proteomics analyses and it is now possible to det. combinatorial patterns of modifications. Presented here is an overview of the most used and newly developed proteomics strategies for histone PTM characterization and a no. of case studies where epigenetic mechanisms have been comprehensively characterized. Moreover, a no. of current epigenetic therapies are illustrated, with an emphasis on cancer.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslCmsLrO&md5=ff9d6a149f9634844059b74831621b1f

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Sidoli, S. ; Garcia, B. A. Middle-down proteomics: a still unexploited resource for chromatin biology Expert Rev. Proteomics 2017 , 14 , 617 – 626 DOI: 10.1080/14789450.2017.1345632

[Crossref], [PubMed], [CAS], Google Scholar

229

Middle-down proteomics: a still unexploited resource for chromatin biology

Sidoli, Simone; Garcia, Benjamin A.

Expert Review of Proteomics (2017), 14 (7), 617-626CODEN: ERPXA3; ISSN:1478-9450. (Taylor & Francis Ltd.)

: Anal. of histone post-translational modifications (PTMs) by mass spectrometry (MS) has become a fundamental tool for the characterization of chromatin compn. and dynamics. Histone PTMs benchmark several biol. states of chromatin, including regions of active enhancers, active/repressed gene promoters and damaged DNA. These complex regulatory mechanisms are often defined by combinatorial histone PTMs; for instance, active enhancers are commonly occupied by both marks H3K4me1 and H3K27ac. The traditional bottom-up MS strategy identifies and quantifies short (aa 4-20) tryptic peptides, and it is thus not suitable for the characterization of combinatorial PTMs.: Here, we review the advancement of the middle-down MS strategy applied to histones, which consists in the anal. of intact histone N-terminal tails (aa 50-60). Middle-down MS has reached sufficient robustness and reliability, and it is far less tech. challenging than PTM quantification on intact histones (top-down). However, the very few chromatin biol. studies applying middle-down MS resulting from PubMed searches indicate that it is still very scarcely exploited, potentially due to the apparent high complexity of method and anal.: We will discuss the state-of-the-art workflow and examples of existing studies, aiming to highlight its potential and feasibility for studies of cell biologists interested in chromatin and epigenetics.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVymsbnO&md5=a331965dfeb3b56c6044cc2d83e88535

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Benevento, M. ; Tonge, P. D. ; Puri, M. C. ; Nagy, A. ; Heck, A. J. R. ; Munoz, J. Proteomics 2015 , 15 , 3219 – 3231 DOI: 10.1002/pmic.201500031

231

Wang, W.-L. ; Anderson, L. C. ; Nicklay, J. J. ; Chen, H. ; Gamble, M. J. ; Shabanowitz, J. ; Hunt, D. F. ; Shechter, D. Epigenet. Chromatin 2014 , 7 , 22 DOI: 10.1186/1756-8935-7-22

[Crossref], [PubMed], [CAS], Google Scholar

231

Phosphorylation and arginine methylation mark histone H2A prior to deposition during Xenopus laevis development

Wang Wei-Lin; Shechter David; Anderson Lissa C; Nicklay Joshua J; Shabanowitz Jeffrey; Chen Hongshan; Gamble Matthew J; Hunt Donald F

Epigenetics & chromatin (2014), 7 (), 22 ISSN:.

BACKGROUND: Stored, soluble histones in eggs are essential for early development, in particular during the maternally controlled early cell cycles in the absence of transcription. Histone post-translational modifications (PTMs) direct and regulate chromatin-templated transactions, so understanding the nature and function of pre-deposition maternal histones is essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Little is known about histone H2A pre-deposition modifications nor known about the transitions that occur upon the onset of zygotic control of the cell cycle and transcription at the mid-blastula transition (MBT). RESULTS: We isolated histones from staged Xenopus laevis oocytes, eggs, embryos, and assembled pronuclei to identify changes in histone H2A modifications prior to deposition and in chromatin. Soluble and chromatin-bound histones from eggs and embryos demonstrated distinct patterns of maternal and zygotic H2A PTMs, with significant pre-deposition quantities of S1ph and R3me1, and R3me2s. We observed the first functional distinction between H2A and H4 S1 phosphorylation, as we showed that H2A and H2A.X-F (also known as H2A.X.3) serine 1 (S1) is phosphorylated concomitant with germinal vesicle breakdown (GVBD) while H4 serine 1 phosphorylation occurs post-MBT. In egg extract H2A/H4 S1 phosphorylation is independent of the cell cycle, chromatin assembly, and DNA replication. H2AS1ph is highly enriched on blastula chromatin during repression of zygotic gene expression while H4S1ph is correlated with the beginning of maternal gene expression and the lengthening of the cell cycle, consistent with distinct biological roles for H2A and H4 S1 phosphorylation. We isolated soluble H2A and H2A.X-F from the egg and chromatin-bound in pronuclei and analyzed them by mass spectrometry analysis to quantitatively determine abundances of S1ph and R3 methylation. We show that H2A and H4 S1ph, R3me1 and R3me2s are enriched on nucleosomes containing both active and repressive histone PTMs in human A549 cells and Xenopus embryos. CONCLUSIONS: Significantly, we demonstrated that H2A phosphorylation and H4 arginine methylation form a new class of bona fide pre-deposition modifications in the vertebrate embryo. We show that S1ph and R3me containing chromatin domains are not correlated with H3 regulatory PTMs, suggesting a unique role for phosphorylation and arginine methylation.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M7ptlGisg%253D%253D&md5=ffd8646fe077d60bab08581d7e4832b3

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Karch, K. R. ; Langelier, M.-F. ; Pascal, J. M. ; Garcia, B. A. Mol. BioSyst. 2017 , 13 , 2660 DOI: 10.1039/C7MB00498B

[Crossref], [PubMed], [CAS], Google Scholar

232

The nucleosomal surface is the main target of histone ADP-ribosylation in response to DNA damage

Karch, Kelly R.; Langelier, Marie-France; Pascal, John M.; Garcia, Benjamin A.

Molecular BioSystems (2017), 13 (12), 2660-2671CODEN: MBOIBW; ISSN:1742-2051. (Royal Society of Chemistry)

ADP-ribosylation is a protein post-translational modification catalyzed by ADP-ribose transferases (ARTs). ART activity is crit. in mediating many cellular processes, and is required for DNA damage repair. All five histone proteins are extensively ADP-ribosylated by ARTs upon induction of DNA damage. However, how these modifications aid in repair processes is largely unknown, primarily due to lack of knowledge about where they site-specifically occur on histones. Here, we conduct a comprehensive anal. of histone Asp/Glu ADP-ribosylation sites upon DNA damage induced by di-Me sulfate (DMS). We also demonstrate that incubation of cell nuclei with NAD+, as has been done previously in the literature, leads to spurious ADP-ribosylation levels of histone proteins. Altogether, we were able to identify 30 modification sites, 20 of which are novel. We also quantify the abundance of these modification sites during the course of DNA damage insult to identify which sites are crit. for mediating repair. We found that every quantifiable site increases in abundance over time and that each identified ADP-ribosylation site is located on the surface of the nucleosome. Together, the data suggest specific Asp/Glu residues are unlikely to be crit. for DNA damage repair and rather that this process is likely dependent on ADP-ribosylation of the nucleosomal surface in general.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1Knu7zK&md5=8d7d35bec3130dd6438fb022674fd1b0

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Olszowy, P. ; Donnelly, M. R. ; Lee, C. ; Ciborowski, P. Proteome Sci. 2015 , 13 , 24 DOI: 10.1186/s12953-015-0080-7

[Crossref], [PubMed], [CAS], Google Scholar

233

Profiling post-translational modifications of histones in human monocyte-derived macrophages

Olszowy, Pawel; Donnelly, Maire Rose; Lee, Chanho; Ciborowski, Pawel

Proteome Science (2015), 13 (), 24/1-24/13CODEN: PSRCCC; ISSN:1477-5956. (BioMed Central Ltd.)

Background: Histones and their post-translational modifications impact cellular function by acting as key regulators in the maintenance and remodeling of chromatin, thus affecting transcription regulation either pos. (activation) or neg. (repression). In this study we describe a comprehensive, bottom-up proteomics approach to profiling post-translational modifications (acetylation, mono-, di- and tri-methylation, phosphorylation, biotinylation, ubiquitination, citrullination and ADP-ribosylation) in human macrophages, which are primary cells of the innate immune system. As our knowledge expands, it becomes more evident that macrophages are a heterogeneous population with potentially subtle differences in their responses to various stimuli driven by highly complex epigenetic regulatory mechanisms. Methods: To profile post-translational modifications (PTMs) of histones in macrophages we used two platforms of liq. chromatog. and mass spectrometry. One platform was based on Sciex5600 TripleTof and the second one was based on VelosPro Orbitrap Elite ETD mass spectrometers. Results: We provide side-by-side comparison of profiling using two mass spectrometric platforms, ion trap and qTOF, coupled with the application of collisional induced and electron transfer dissocn. We show for the first time methylation of a His residue in macrophages and demonstrate differences in histone PTMs between those currently reported for macrophage cell lines and what we identified in primary cells. We have found a relatively low level of histone PTMs in differentiated but resting human primary monocyte derived macrophages. Conclusions: This study is the first comprehensive profiling of histone PTMs in primary human MDM. Our study implies that epigenetic regulatory mechanisms operative in transformed cell lines and primary cells are overlapping to a limited extent. Our mass spectrometric approach provides groundwork for the investigation of how histone PTMs contribute to epigenetic regulation in primary human macrophages.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtVCltLo%253D&md5=2e40c4d4008b2e5969534f248fd20629

234

Schräder, C. U. ; Lee, L. ; Rey, M. ; Sarpe, V. ; Man, P. ; Sharma, S. ; Zabrouskov, V. ; Larsen, B. ; Schriemer, D. C. Mol. Cell. Proteomics 2017 , 16 , 1162 – 1171 DOI: 10.1074/mcp.M116.066803

[Crossref], [PubMed], [CAS], Google Scholar

234

Neprosin, a Selective Prolyl Endoprotease for Bottom-up Proteomics and Histone Mapping

Schrader, Christoph U.; Lee, Linda; Rey, Martial; Sarpe, Vladimir; Man, Petr; Sharma, Seema; Zabrouskov, Vlad; Larsen, Brett; Schriemer, David C.

Molecular & Cellular Proteomics (2017), 16 (6), 1162-1171CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Addnl., enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate.". Digestion proceeds to a stable end point, resulting in an av. peptide mass of 2521 units and a higher dependence upon electron-transfer dissocn. for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high av. peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the anal. of co-occurring post-translational modifications in these important N-terminal tails.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXptFCntrk%253D&md5=2c24fac4fc12e63189cdf7b26f5b6e58

235

Sidoli, S. ; Schwämmle, V. ; Ruminowicz, C. ; Hansen, T. A. ; Wu, X. ; Helin, K. ; Jensen, O. N. Proteomics 2014 , 14 , 2200 – 2211 DOI: 10.1002/pmic.201400084

[Crossref], [PubMed], [CAS], Google Scholar

235

Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones

Sidoli, Simone; Schwaemmle, Veit; Ruminowicz, Chrystian; Hansen, Thomas A.; Wu, Xudong; Helin, Kristian; Jensen, Ole N.

Proteomics (2014), 14 (19), 2200-2211CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)

We present an integrated middle-down proteomics platform for sensitive mapping and quantification of coexisting PTMs in large polypeptides (5-7 kDa). We combined an RP trap column with subsequent weak cation exchange-hydrophilic interaction LC interfaced directly to high mass accuracy ESI MS/MS using electron transfer dissocn. This enabled automated and efficient sepn. and sequencing of hypermodified histone N-terminal tails for unambiguous localization of combinatorial PTMs. We present Histone Coder and IsoScale software to ext., filter, and analyze MS/MS data, including quantification of cofragmenting isobaric polypeptide species. We characterized histone tails derived from murine embryonic stem cells knockout in suppressor of zeste12 (Suz12-/-) and quantified 256 combinatorial histone marks in histones H3, H4, and H2A. Furthermore, a total of 713 different combinatorial histone marks were identified in purified histone H3. We measured a seven-fold redn. of H3K27me2/me3 (where me2 and me3 are dimethylation and trimethylation, resp.) in Suz12-/- cells and detected significant changes of the relative abundance of 16 other single PTMs of histone H3 and other combinatorial marks. We conclude that the inactivation of Suz12 is assocd. with changes in the abundance of not only H3K27 methylation but also multiple other PTMs in histone H3 tails.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVersrnE&md5=1bbdf43242718320e77d5e398f1c7555

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Tian, Z. ; Tolić, N. ; Zhao, R. ; Moore, R. J. ; Hengel, S. M. ; Robinson, E. W. ; Stenoien, D. L. ; Wu, S. ; Smith, R. D. ; Paša-Tolić, L. Genome Biol. 2012 , 13 , r86 DOI: 10.1186/gb-2012-13-10-r86

[Crossref], [PubMed], [CAS], Google Scholar

236

Enhanced top-down characterization of histone post-translational modifications

Tian, Zhixin; Tolic, Nikola; Zhao, Rui; Moore, Ronald J.; Hengel, Shawna M.; Robinson, Errol W.; Stenoien, David L.; Wu, Si; Smith, Richard D.; Pasa-Tolic, Ljiljana

Genome Biology (2012), 13 (), R86CODEN: GNBLFW; ISSN:1474-760X. (BioMed Central Ltd.)

Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liq. chromatog.-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS anal. of 7.5 μg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVKjtrjP&md5=9f4120bb29c95543d76c1c7e608ef437

237

Faserl, K. ; Sarg, B. ; Maurer, V. ; Lindner, H. H. J. Chromatogr. A 2017 , 1498 , 215 – 223 DOI: 10.1016/j.chroma.2017.01.086

238

Shliaha, P. V. ; Baird, M. A. ; Nielsen, M. M. ; Gorshkov, V. ; Bowman, A. P. ; Kaszycki, J. L. ; Jensen, O. N. ; Shvartsburg, A. A. Anal. Chem. 2017 , 89 , 5461 – 5466 DOI: 10.1021/acs.analchem.7b00379

[ACS Full Text ], [CAS], Google Scholar

238

Characterization of Complete Histone Tail Proteoforms Using Differential Ion Mobility Spectrometry

Shliaha, Pavel V.; Baird, Matthew A.; Nielsen, Mogens M.; Gorshkov, Vladimir; Bowman, Andrew P.; Kaszycki, Julia L.; Jensen, Ole N.; Shvartsburg, Alexandre A.

Analytical Chemistry (Washington, DC, United States) (2017), 89 (10), 5461-5466CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Histone proteins are subject to dynamic posttranslational modifications (PTMs) that cooperatively modulate the chromatin structure and function. Nearly all functional PTMs are found on the N-terminal histone domains (tails) of ∼50 residues protruding from the nucleosome core. Using high-definition differential ion mobility spectrometry (FAIMS) with electron transfer dissocn., the authors demonstrate rapid baseline gas-phase sepn. and identification of tails involving monomethylation, trimethylation, acetylation, or phosphorylation in biol. relevant positions. These are by far the largest variant peptides resolved by any method, some with PTM contributing just 0.25% to the mass. This opens the door to similar sepns. for intact proteins and in top-down proteomics.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlvF2ku7c%253D&md5=ca09c13a1b926b2a17af9a160444a0ac

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Sweredoski, M. J. ; Pekar Second, T. ; Broeker, J. ; Moradian, A. ; Hess, S. Int. J. Mass Spectrom. 2015 , 390 , 155 – 162 DOI: 10.1016/j.ijms.2015.06.018

240

Sidoli, S. ; Lin, S. ; Karch, K. R. ; Garcia, B. A. Anal. Chem. 2015 , 87 , 3129 – 3133 DOI: 10.1021/acs.analchem.5b00072

[ACS Full Text ], [CAS], Google Scholar

240

Bottom-Up and Middle-Down Proteomics Have Comparable Accuracies in Defining Histone Post-Translational Modification Relative Abundance and Stoichiometry

Sidoli, Simone; Lin, Shu; Karch, Kelly R.; Garcia, Benjamin A.

Analytical Chemistry (Washington, DC, United States) (2015), 87 (6), 3129-3133CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Histone proteins are key components of chromatin. Their N-terminal tails are enriched in combinatorial post-translational modifications (PTMs), which influence gene regulation, DNA repair, and chromosome condensation. Mass spectrometry (MS)-based middle-down proteomics has emerged as a technique to analyze cooccurring PTMs, as it allows for the characterization of intact histone tails (>50 aa) rather than short (<20 aa) peptides analyzed by bottom-up. However, a demonstration of its reliability is still lacking. The authors compared results obtained with the middle-down and the bottom-up strategy in calcg. PTM relative abundance and stoichiometry. Since bottom-up was proven to have biases in peptide signal detection such as uneven ionization efficiency, the authors performed an external correction using a synthetic peptide library with known peptide relative abundance. Cor. bottom-up data were used as ref. Calcd. abundances of single PTMs showed similar deviations from the ref. when comparing middle-down and uncorrected bottom-up results. Moreover, the two strategies provided similar performance in defining accurate PTM stoichiometry. Collectively, the authors evidenced that the middle-down strategy is at least equally reliable to bottom-up in quantifying histone PTMs.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjsFCisL4%253D&md5=6657b2c6adb32db9c89ae827e6f83256

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Sidoli, S. ; Lu, C. ; Coradin, M. ; Wang, X. ; Karch, K. R. ; Ruminowicz, C. ; Garcia, B. A. Epigenet. Chromatin 2017 , 10 , 34 DOI: 10.1186/s13072-017-0139-z

[Crossref], [PubMed], [CAS], Google Scholar

241

Metabolic labeling in middle-down proteomics allows for investigation of the dynamics of the histone code

Sidoli Simone; Lu Congcong; Coradin Mariel; Wang Xiaoshi; Karch Kelly R; Garcia Benjamin A; Ruminowicz Chrystian

Epigenetics & chromatin (2017), 10 (1), 34 ISSN:.

BACKGROUND: Middle-down mass spectrometry (MS), i.e., analysis of long (~50-60 aa) polypeptides, has become the method with the highest throughput and accuracy for the characterization of combinatorial histone posttranslational modifications (PTMs). The discovery of histone readers with multiple domains, and overall the cross talk of PTMs that decorate histone proteins, has revealed that histone marks have synergistic roles in modulating enzyme recruitment and subsequent chromatin activities. Here, we demonstrate that the middle-down MS strategy can be combined with metabolic labeling for enhanced quantification of histone proteins and their combinatorial PTMs in a dynamic manner. METHODS: We used a nanoHPLC-MS/MS system consisting of hybrid weak cation exchange-hydrophilic interaction chromatography combined with high resolution MS and MS/MS with ETD fragmentation. After spectra identification, we filtered confident hits and quantified polypeptides using our in-house software isoScale. RESULTS: We first verified that middle-down MS can discriminate and differentially quantify unlabeled from heavy labeled histone N-terminal tails (heavy lysine and arginine residues). Results revealed no bias toward identifying and quantifying unlabeled versus heavy labeled tails, even if the heavy labeled peptides presented the typical skewed isotopic pattern typical of long protein sequences that hardly get 100% labeling. Next, we plated epithelial cells into a media with heavy methionine-(methyl-(13)CD3), the precursor of the methyl donor S-adenosylmethionine and stimulated epithelial to mesenchymal transition (EMT). We assessed that results were reproducible across biological replicates and with data obtained using the more widely adopted bottom-up MS strategy, i.e., analysis of short tryptic peptides. We found remarkable differences in the incorporation rate of methylations in non-confluent cells versus confluent cells. Moreover, we showed that H3K27me3 was a critical player during the EMT process, as a consistent portion of histones modified as H3K27me2K36me2 in epithelial cells were converted into H3K27me3K36me2 in mesenchymal cells. CONCLUSIONS: We demonstrate that middle-down MS, despite being a more scarcely exploited MS technique than bottom-up, is a robust quantitative method for histone PTM characterization. In particular, middle-down MS combined with metabolic labeling is currently the only methodology available for investigating turnover of combinatorial histone PTMs in dynamic systems.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cjltFyhtA%253D%253D&md5=9bfa1a97451e76177c6cdc4f171ca1fd

242

Sweredoski, M. J. ; Moradian, A. ; Raedle, M. ; Franco, C. ; Hess, S. Anal. Chem. 2015 , 87 , 8360 – 8366 DOI: 10.1021/acs.analchem.5b01542

[ACS Full Text ], [CAS], Google Scholar

242

High Resolution Parallel Reaction Monitoring with Electron Transfer Dissociation for Middle-Down Proteomics

Sweredoski, Michael J.; Moradian, Annie; Raedle, Matthias; Franco, Catarina; Hess, Sonja

Analytical Chemistry (Washington, DC, United States) (2015), 87 (16), 8360-8366CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

In recent years, middle-down proteomics has emerged as a popular technique for the characterization and quantification of proteins not readily amenable to typical bottom-up approaches. So far, all high resoln. middle-down approaches are done in data-dependent acquisition mode, using both collision-induced dissocn. or electron capture/transfer dissocn. techniques. Here, we explore middle-down proteomics with electron transfer dissocn. using a targeted acquisition mode, parallel reaction monitoring (PRM), on an Orbitrap Fusion. As an example of a highly modified protein, we used histone H3 fractions from untreated and DMSO-treated Murine ErythroLeukemia (MEL) cells. We first detd. optimized instrument parameters to obtain high sequence coverage using a synthetic std. peptide. We then setup a combined method of both MS1 scans and PRM scans of the 20 most abundant combinations of methylation and acetylation of the +10 charge state of the N-terminal tail of H3. Weak cation exchange hydrophilic interaction chromatog. was used to sep. the N-terminal H3 tail, primarily, by its acetylation and, to a secondary degree, by its methylation status, which aided in the interpretation of the results. After deconvolution of the highly charged ions, peaks were annotated to a min. set of 254 H3 proteoforms in the untreated and treated samples. Upon DMSO treatment, global quantitation changes from the MS1 level show a relative decrease of 2, 3, 4, and 5 acetylations and an increase of 0 and 1 acetylations. A fragment ion map was developed to visualize specific differences between treated and untreated samples. Taken together, the data presented here show that middle-down proteomics with electron transfer dissocn. using PRM is a novel, attractive method for the effective anal. and quantification of large and highly modified peptides.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFOms7rP&md5=3f3fadb562ec99bf497232c626da1604

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Dang, X. ; Scotcher, J. ; Wu, S. ; Chu, R. K. ; Tolić, N. ; Ntai, I. ; Thomas, P. M. ; Fellers, R. T. ; Early, B. P. ; Zheng, Y. ; Durbin, K. R. ; LeDuc, R. D. ; Wolff, J. J. ; Thompson, C. J. ; Pan, J. ; Han, J. ; Shaw, J. B. ; Salisbury, J. P. ; Easterling, M. ; Borchers, C. H. ; Brodbelt, J. S. ; Agar, J. N. ; Paša-Tolić, L. ; Kelleher, N. L. ; Young, N. L. Proteomics 2014 , 14 , 1130 – 1140 DOI: 10.1002/pmic.201300438

[Crossref], [PubMed], [CAS], Google Scholar

243

The first pilot project of the consortium for top-down proteomics: A status report

Dang, Xibei; Scotcher, Jenna; Wu, Si; Chu, Rosalie K.; Tolic, Nikola; Ntai, Ioanna; Thomas, Paul M.; Fellers, Ryan T.; Early, Bryan P.; Zheng, Yupeng; Durbin, Kenneth R.; LeDuc, Richard D.; Wolff, Jeremy J.; Thompson, Christopher J.; Pan, Jingxi; Han, Jun; Shaw, Jared B.; Salisbury, Joseph P.; Easterling, Michael; Borchers, Christoph H.; Brodbelt, Jennifer S.; Agar, Jeffery N.; Pasa-Tolic, Ljiljana; Kelleher, Neil L.; Young, Nicolas L.

Proteomics (2014), 14 (10), 1130-1140CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)

Pilot Project #1-the identification and characterization of human histone H4 proteoforms by top-down MS-is the first project launched by the Consortium for Top-Down Proteomics (CTDP) to refine and validate top-down MS. Within the initial results from seven participating labs., all reported the probability-based identification of human histone H4 (UniProt accession P62805) with expectation values ranging from 10-13 to 10-105. Regarding characterization, a total of 74 proteoforms were reported, with 21 done so unambiguously; one new PTM, K79ac, was identified. Inter-lab. comparison reveals aspects of the results that are consistent, such as the localization of individual PTMs and binary combinations, while other aspects are more variable, such as the accurate characterization of low-abundance proteoforms harboring >2 PTMs. An open-access tool and discussion of proteoform scoring are included, along with a description of general challenges that lie ahead including improved proteoform sepns. prior to mass spectrometric anal., better instrumentation performance, and software development.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtVCqtL4%253D&md5=f358d3c21e5957c7d55b60c72a2b30a9

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Dang, X. ; Singh, A. ; Spetman, B. D. ; Nolan, K. D. ; Isaacs, J. S. ; Dennis, J. H. ; Dalton, S. ; Marshall, A. G. ; Young, N. L. J. Proteome Res. 2016 , 15 , 3196 – 3203 DOI: 10.1021/acs.jproteome.6b00414

[ACS Full Text ], [CAS], Google Scholar

244

Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS

Dang, Xibei; Singh, Amar; Spetman, Brian D.; Nolan, Krystal D.; Isaacs, Jennifer S.; Dennis, Jonathan H.; Dalton, Stephen; Marshall, Alan G.; Young, Nicolas L.

Journal of Proteome Research (2016), 15 (9), 3196-3203CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Histone variants are known to play a central role in genome regulation and maintenance. However, many variants are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry due to their highly similar sequences. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. Here, ultra-high-resoln. FT-ICR MS and MS/MS yield quant. relative abundances of all detected HeLa H2A and H2B isobaric and isomeric variants with a label-free approach. We extend the anal. to identify and relatively quantitate 16 proteoforms from 12 sequence variants of histone H2A and 10 proteoforms of histone H2B from three other cell lines: human embryonic stem cells (WA09), U937, and a prostate cancer cell line LaZ. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biol. role of many previously unstudied histone variants and post-translational modifications.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFOktLbN&md5=7c9988f6884c1f7d17033c7341682e5f

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Anderson, L. C. ; Karch, K. R. ; Ugrin, S. A. ; Coradin, M. ; English, A. M. ; Sidoli, S. ; Shabanowitz, J. ; Garcia, B. A. ; Hunt, D. F. Mol. Cell. Proteomics 2016 , 15 , 975 – 988 DOI: 10.1074/mcp.O115.053843

[Crossref], [PubMed], [CAS], Google Scholar

245

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Anderson, Lissa C.; Karch, Kelly R.; Ugrin, Scott A.; Coradin, Mariel; English, A. Michelle; Sidoli, Simone; Shabanowitz, Jeffrey; Garcia, Benjamin A.; Hunt, Donald F.

Molecular & Cellular Proteomics (2016), 15 (3), 975-988CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Histones represent a class of proteins ideally suited to analyses by top-down mass spectrometry due to their relatively small size, the high electron transfer dissocn.-compatible charge states they exhibit, and the potential to gain valuable information concerning combinatorial post-translational modifications and variants. Here, we report an extension of these techniques. Sequential ion/ion reactions carried out in a modified Orbitrap Velos Pro/EliteTM capable of multiple fragment ion fills of the C-trap, in combination with data-dependent and targeted HPLC-MS expts., were used to obtain high resoln. MS/MS spectra of histones from butyrate-treated HeLa cells. These spectra were used to identify several unique intact histone proteoforms with up to 81% sequence coverage. We also demonstrate that parallel ion parking during ion/ion proton transfer reactions can be used to sep. species of overlapping m/z that are not sepd. chromatog., revealing previously indiscernible signals. Finally, we characterized several truncated forms of H2A and H2B found within the histone fractions analyzed, achieving up to 93% sequence coverage by electron transfer dissocn. MS/MS. Results of follow-up in vitro expts. suggest that some of the truncated histone H2A proteoforms we obsd. can be generated by cathepsin L, an enzyme known to also catalyze clipping of histone H3.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjsVGruro%253D&md5=ec7d76ab4c9ae32735517029acf4f436

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Zheng, Y. ; Fornelli, L. ; Compton, P. D. ; Sharma, S. ; Canterbury, J. ; Mullen, C. ; Zabrouskov, V. ; Fellers, R. T. ; Thomas, P. M. ; Licht, J. D. ; Senko, M. W. ; Kelleher, N. L. Mol. Cell. Proteomics 2016 , 15 , 776 – 790 DOI: 10.1074/mcp.M115.053819

[Crossref], [PubMed], [CAS], Google Scholar

246

Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression

Zheng, Yupeng; Fornelli, Luca; Compton, Philip D.; Sharma, Seema; Canterbury, Jesse; Mullen, Christopher; Zabrouskov, Vlad; Fellers, Ryan T.; Thomas, Paul M.; Licht, Jonathan D.; Senko, Michael W.; Kelleher, Neil L.

Molecular & Cellular Proteomics (2016), 15 (3), 776-790CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Histones, and their modifications, are crit. components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technol. for clean isolation of isobaric proteoforms contg. up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential anal. of modifications by electron-based dissocn. recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a min. of three Me groups in H3K9 and H3K27 suggested a hierarchy in the addn. of certain modifications. Comparative anal. showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms contg. H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such "trivalent hypermethylation" (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry can now decode crosstalk involving up to three modified sites.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjsVGru7k%253D&md5=ae544664ce6c3fee385278de39a10136

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Molden, R. C. ; Bhanu, N. V. ; LeRoy, G. ; Arnaudo, A. M. ; Garcia, B. A. Epigenet. Chromatin 2015 , 8 , 15 DOI: 10.1186/s13072-015-0006-8

[Crossref], [PubMed], [CAS], Google Scholar

247

Multi-faceted quantitative proteomics analysis of histone H2B isoforms and their modifications

Molden Rosalynn C; LeRoy Gary; Bhanu Natarajan V; Garcia Benjamin A; Arnaudo Anna M

Epigenetics & chromatin (2015), 8 (), 15 ISSN:.

BACKGROUND: Histone isoforms and their post-translational modifications (PTMs) play an important role in the control of many chromatin-related processes including transcription and DNA damage. Variants of histones H2A and H3 have been studied in depth and have been found to have distinct functions. Although 13 somatic histone H2B isoforms have been identified by various biochemical and mass spectrometric (MS) approaches, the distinct roles of these isoforms within human cells are as yet unknown. Here, we have developed quantitative MS techniques to characterize isoform-specific H2B expression across the cell cycle, in differentiated myogenic cells, and in different cancer cell lines to illuminate potential functional roles. RESULTS: Using the MS strategies that we developed, we identified differences in H2B isoform levels between different cancer cell types, suggesting cancer or tissue-specific H2B isoform regulation. In particular, we found large variations in the levels of isoforms H2B1B and H2B1M across the panel of cell lines. We also found that, while individual H2B isoforms do not differ in their acetylation levels, trends in the acetylation on all H2B isoforms correlated with acetylation on other histone family members in the cancer cell line panel. We also used the MS strategies to study H2B protein expression across the cell cycle and determined that H2B isoforms that are alternatively spliced to carry a polyadenylation signal rather than the standard histone downstream element are expressed independently of the cell cycle. However, the level of protein produced from the polyadenylated transcripts does not contribute significantly to the total pool of H2B isoforms translated across the cell cycle or in non-cycling myogenic cells. CONCLUSIONS: Our results show that H2B isoforms are expressed at varying levels in different cells, suggesting isoform-specific, and possibly cell-type-specific, H2B gene regulation. The bottom-up mass spectrometry technique we developed for H2B quantification is compatible with the current standard histone H3 and H4 bottom-up 'one-pot' analysis platform so that H2B isoforms and their modifications can be studied in future experiments at the same time as histone H3 and H4 modifications. Therefore, we have expanded the histone landscape that can be interrogated in future experiments.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MjovFegsg%253D%253D&md5=6a8fa5f07de5a7224805acb51181cebf

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Zhang, H. ; Cui, W. ; Gross, M. L. FEBS Lett. 2014 , 588 , 308 – 317 DOI: 10.1016/j.febslet.2013.11.027

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Guthals, A. ; Gan, Y. ; Murray, L. ; Chen, Y. ; Stinson, J. ; Nakamura, G. ; Lill, J. R. ; Sandoval, W. ; Bandeira, N. J. Proteome Res. 2017 , 16 , 45 – 54 DOI: 10.1021/acs.jproteome.6b00608

[ACS Full Text ], [CAS], Google Scholar

249

De Novo MS/MS Sequencing of Native Human Antibodies

Guthals, Adrian; Gan, Yutian; Murray, Laura; Chen, Yongmei; Stinson, Jeremy; Nakamura, Gerald; Lill, Jennie R.; Sandoval, Wendy; Bandeira, Nuno

Journal of Proteome Research (2017), 16 (1), 45-54CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

One direct route for the discovery of therapeutic human monoclonal antibodies (mAbs) involves the isolation of peripheral B cells from survivors/sero-pos. individuals after exposure to an infectious reagent or disease etiol., followed by single-cell sequencing or hybridoma generation. Peripheral B cells, however, are not always easy to obtain and represent only a small percentage of the total B-cell population across all bodily tissues. Although it has been demonstrated that tandem mass spectrometry (MS/MS) techniques can interrogate the full polyclonal antibody (pAb) response to an antigen in vivo, all current approaches identify MS/MS spectra against databases derived from genetic sequencing of B cells from the same patient. In this proof-of-concept study, the authors demonstrate the feasibility of a novel MS/MS antibody discovery approach in which only serum antibodies are required without the need for sequencing of genetic material. Peripheral pAbs from a cytomegalovirus-exposed individual were purified by glycoprotein B antigen affinity and de novo sequenced from MS/MS data. Purely MS-derived mAbs were then manufd. in mammalian cells to validate potency via antigen-binding ELISA. Interestingly, the authors found that these mAbs accounted for 1 to 2% of total donor IgG but were not detected in parallel sequencing of memory B cells from the same patient.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhslamt7jE&md5=15b6d2ef8b7438fd6fc4da61790d58a7

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Pang, Y. ; Wang, W.-H. ; Reid, G. E. ; Hunt, D. F. ; Bruening, M. L. Anal. Chem. 2015 , 87 , 10942 – 10949 DOI: 10.1021/acs.analchem.5b02739

[ACS Full Text ], [CAS], Google Scholar

250

Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis

Pang, Yongle; Wang, Wei-Han; Reid, Gavin E.; Hunt, Donald F.; Bruening, Merlin L.

Analytical Chemistry (Washington, DC, United States) (2015), 87 (21), 10942-10949CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Monoclonal antibodies (mAbs) are the fastest growing class of therapeutic drugs, because of their high specificities to target cells. Facile anal. of therapeutic mAbs and their post-translational modifications (PTMs) is essential for quality control, and mass spectrometry (MS) is the most powerful tool for antibody characterization. This study uses pepsin-contg. nylon membranes as controlled proteolysis reactors for mAb digestion prior to ultrahigh-resoln. Orbitrap MS anal. Variation of the residence times (from 3 ms to 3 s) of antibody solns. in the membranes yields "bottom-up" (1-2 kDa) to "middle-down" (5-15 kDa) peptide sizes within less than 10 min. These peptides cover the entire sequences of Trastuzumab and a Waters antibody, and a proteolytic peptide comprised of 140 amino acids from the Waters antibody contains all three complementarity detg. regions on the light chain. This work compares the performance of "bottom-up" (in-soln. tryptic digestion), "top-down" (intact protein fragmentation), and "middle-down" (in-membrane digestion) anal. of an antibody light chain. Data from tandem MS show 99%, 55%, and 99% bond cleavage for "bottom-up", "top-down", and "middle-down" analyses, resp. In-membrane digestion also facilitates detection of PTMs such as oxidn., deamidation, N-terminal pyroglutamic acid formation, and glycosylation. Compared to "bottom-up" and "top-down" approaches for antibody characterization, in-membrane digestion uses minimal sample prepn. time, and this technique also yields high peptide and sequence coverage for the identification of PTMs.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1Chtr%252FO&md5=e6f01a3af1eb4ed5ea4540cf8525db3c

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Zhang, L. ; English, A. M. ; Bai, D. L. ; Ugrin, S. A. ; Shabanowitz, J. ; Ross, M. M. ; Hunt, D. F. ; Wang, W.-H. Mol. Cell. Proteomics 2016 , 15 , 1479 – 1488 DOI: 10.1074/mcp.O115.056721

[Crossref], [PubMed], [CAS], Google Scholar

251

Analysis of Monoclonal Antibody Sequence and Post-translational Modifications by Time-controlled Proteolysis and Tandem Mass Spectrometry

Zhang, Lichao; English, A. Michelle; Bai, Dina L.; Ugrin, Scott A.; Shabanowitz, Jeffrey; Ross, Mark M.; Hunt, Donald F.; Wang, Wei-Han

Molecular & Cellular Proteomics (2016), 15 (4), 1479-1488CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Methodol. for sequence anal. of ∼150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8 m urea at a controlled flow rate through a micro column reactor contg. immobilized aspergillopepsin I. The resulting product mixt. contg. 3-9 kDa peptides was then fractionated by capillary column liq. chromatog. and analyzed online by both electron-transfer dissocn. and collisionally activated dissocn. mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous post-translational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms of N-linked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide contg. peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the mol. requires longer digestion times. The identity of two peptides connected by a disulfide bond is detd. using a combination of electron-transfer dissocn. and ion-ion proton transfer chem. to read the two N-terminal and two C-terminal sequences of the connected peptides.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xltlansbg%253D&md5=5b82931074d16fcb1864c4a998b56121

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Pan, J. ; Zhang, S. ; Chou, A. ; Borchers, C. H. Chem. Sci. 2016 , 7 , 1480 – 1486 DOI: 10.1039/C5SC03420E

[Crossref], [CAS], Google Scholar

252

Higher-order structural interrogation of antibodies using middle-down hydrogen/deuterium exchange mass spectrometry

Pan, Jingxi; Zhang, Suping; Chou, Albert; Borchers, Christoph H.

Chemical Science (2016), 7 (2), 1480-1486CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)

Although X-ray crystallog. is the "gold std." method for protein higher-order structure anal., the challenges of antibody crystn. and the time-consuming data anal. involved make this technique unsuitable for routine structural studies of antibodies. In addn., crystallog. cannot be used for the structural characterization of an antibody in soln., under conditions where antibody drugs are active. Intact antibodies are also too large and too complex for NMR. Top-down mass spectrometry coupled to hydrogen/deuterium exchange (HDX) is a powerful tool for high-resoln. protein structural characterization, but its success declines rapidly as protein size increases. Here we report for the first time a new hybrid "middle-down" HDX approach that overcomes this limitation through enabling the nonspecific enzyme pepsin to perform specific restricted digestion at low pH prior to HPLC sepn. at subzero temps. and online electron transfer dissocn. (ETD). Three large specific peptic fragments (12 to 25 kDa) were obsd. from the heavy chain and light chain of a therapeutic antibody Herceptin, together with a few smaller fragments from the middle portion of the heavy chain. The av. amino-acid resolns. obtained by ETD were around two residues, with single-residue resoln. in many regions. This middle-down approach is also applicable to other antibodies. It provided HDX information on the entire light chain, and 95.3% of the heavy chain, representing 96.8% of the entire antibody (150 kDa). The structural effects of glycosylation on Herceptin were detd. at close-to-single residue level by this method.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFWltr3J&md5=28cd4ae2eeb645801cb3c66d746887fa

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Srzentić, K. ; Fornelli, L. ; Laskay, Ü. A. ; Monod, M. ; Beck, A. ; Ayoub, D. ; Tsybin, Y. O. Anal. Chem. 2014 , 86 , 9945 – 9953 DOI: 10.1021/ac502766n

[ACS Full Text ], [CAS], Google Scholar

253

Advantages of Extended Bottom-Up Proteomics Using Sap9 for Analysis of Monoclonal Antibodies

Srzentic, Kristina; Fornelli, Luca; Laskay, Unige A.; Monod, Michel; Beck, Alain; Ayoub, Daniel; Tsybin, Yury O.

Analytical Chemistry (Washington, DC, United States) (2014), 86 (19), 9945-9953CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Despite the recent advances in structural anal. of monoclonal antibodies with bottom-up, middle-down, and top-down mass spectrometry (MS), further improvements in anal. accuracy, depth, and speed are needed. The remaining challenges include quant. accurate assignment of post-translational modifications, redn. of artifacts introduced during sample prepn., increased sequence coverage per liq. chromatog. (LC) MS expt., and ability to extend the detailed characterization to simple antibody cocktails and more complex antibody mixts. Here, we evaluate the recently introduced extended bottom-up proteomics (eBUP) approach based on proteolysis with secreted aspartic protease 9, Sap9, for anal. of monoclonal antibodies. Key findings of the Sap9-based proteomics anal. of a single antibody include: (i) extensive antibody sequence coverage with up to 100% for the light chain and up to 99-100% for the heavy chain in a single LC-MS run; (ii) connectivity of complementarity-detg. regions (CDRs) via Sap9-produced large proteolytic peptides (3.4 kDa on av.) contg. up to two CDRs per peptide; (iii) reduced artifact introduction (e. g., deamidation) during proteolysis with Sap9 compared to conventional bottom-up proteomics workflows. The anal. of a mixt. of six antibodies via Sap9-based eBUP produced comparable results. Due to the reasons specified above, Sap9-produced proteolytic peptides improve the identification confidence of antibodies from the mixts. compared to conventional bottom-up proteomics dealing with shorter proteolytic peptides.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFamsr%252FK&md5=b24e931cb352c7b8046ef3394097c452

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Fornelli, L. ; Ayoub, D. ; Aizikov, K. ; Beck, A. ; Tsybin, Y. O. Anal. Chem. 2014 , 86 , 3005 – 3012 DOI: 10.1021/ac4036857

[ACS Full Text ], [CAS], Google Scholar

254

Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry

Fornelli, Luca; Ayoub, Daniel; Aizikov, Konstantin; Beck, Alain; Tsybin, Yury O.

Analytical Chemistry (Washington, DC, United States) (2014), 86 (6), 3005-3012CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

The rapid growth of approved biotherapeutics, e.g., monoclonal antibodies or Igs G (IgGs), demands improved techniques for their quality control. Traditionally, proteolysis-based bottom-up mass spectrometry (MS) has been employed. However, the long, multistep sample prepn. protocols required for bottom-up MS are known to potentially introduce artifacts in the original sample. For this reason, a top-down MS approach would be preferable. The current performance of top-down MS of intact monoclonal IgGs, though, enables reaching only up to ∼30% sequence coverage, with incomplete sequencing of the complementarity detg. regions which are fundamental for IgG's antigen binding. Here, the authors describe a middle-down MS protocol based on the use of IgG-degrading enzyme of Streptococcus pyogenes (IdeS), which is capable of digesting IgGs in only 30 min. After chem. redn., the obtained ∼25 kDa proteolytic fragments were analyzed by reversed phase liq. chromatog. (LC) coupled online with an electron transfer dissocn. (ETD)-enabled hybrid Orbitrap Fourier transform mass spectrometer (Orbitrap Elite FTMS). Upon optimization of ETD and product ion transfer parameters, results show that up to ∼50% sequence coverage for selected IgG fragments is reached in a single LC run and up to ∼70% when data obtained by distinct LC-MS runs are averaged. Importantly, the authors demonstrate the potential of this middle-down approach in the identification of oxidized methionine residues. The described approach shows a particular potential for the anal. of IgG mixts.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXis1egtr4%253D&md5=04691eae785cfbb3814e4c6dcf28dcea

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Tran, B. Q. ; Barton, C. ; Feng, J. ; Sandjong, A. ; Yoon, S. H. ; Awasthi, S. ; Liang, T. ; Khan, M. M. ; Kilgour, D. P. A. ; Goodlett, D. R. ; Goo, Y. A. J. Proteomics 2016 , 134 , 93 – 101 DOI: 10.1016/j.jprot.2015.10.021

[Crossref], [PubMed], [CAS], Google Scholar

255

Comprehensive glycosylation profiling of IgG and IgG-fusion proteins by top-down MS with multiple fragmentation techniques

Tran, Bao Quoc; Barton, Christopher; Feng, Jinhua; Sandjong, Aimee; Yoon, Sung Hwan; Awasthi, Shivangi; Liang, Tao; Khan, Mohd. M.; Kilgour, David P. A.; Goodlett, David R.; Goo, Young Ah

Journal of Proteomics (2016), 134 (), 93-101CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)

We employed top- and middle-down analyses with multiple fragmentation techniques including electron transfer dissocn. (ETD), electron capture dissocn. (ECD), and matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) for characterization of a ref. monoclonal antibody (mAb) IgG1 and a fusion IgG protein. Fourier transform ion cyclotron resonance (FT-ICR) or high performance liq. chromatog. electrospray ionization (HPLC-ESI) on an Orbitrap was employed. These expts. provided a comprehensive view on the protein species; esp. for different glycosylation level in these two proteins, which showed good agreement with oligosaccharide profiling. Top- and middle-down MS provided addnl. information regarding glycosylation sites and different combinational protein species that were not available from oligosaccharide mapping or conventional bottom-up anal. Finally, incorporating a limited enzymic digestion by IgG-degrading enzyme of Streptococcus pyogene (IdeS) with MALDI-ISD anal. enabled extended sequence coverage of the internal region of protein without pre-fractionation. Oligosaccharide profiling together with top- and middle-down methods enabled: 1) detection of heterogeneous glycosylated protein species and sites in intact IgG1 and fusion proteins with high mass accuracy, 2) estn. of relative abundance levels of protein species in the sample, 3) confirmation of the protein termini structural information, and 4) improved sequence coverage by MALDI-ISD anal. for the internal regions of the proteins without sample pre-fractionation.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslekt73J&md5=36c51391e599b4f62e2a6f42d7aba19c

256

Jensen, P. F. ; Larraillet, V. ; Schlothauer, T. ; Kettenberger, H. ; Hilger, M. ; Rand, K. D. Mol. Cell. Proteomics 2015 , 14 , 148 – 161 DOI: 10.1074/mcp.M114.042044

[Crossref], [PubMed], [CAS], Google Scholar

256

Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman; Kettenberger, Hubert; Hilger, Maximiliane; Rand, Kasper D.

Molecular & Cellular Proteomics (2015), 14 (1), 148-161CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

The recycling of Igs by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissocn. to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallog. studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG-FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as obsd. for the wild-type glycosylated IgG. Our results provide new mol. insight into the IgG-FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in soln.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXktFGjsg%253D%253D&md5=eba1c63e17f6f3b898c4b13b5458ed8d

257

He, L. ; Anderson, L. C. ; Barnidge, D. R. ; Murray, D. L. ; Hendrickson, C. L. ; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2017 , 28 , 827 – 838 DOI: 10.1007/s13361-017-1602-6

[Crossref], [PubMed], [CAS], Google Scholar

257

Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

He, Lidong; Anderson, Lissa C.; Barnidge, David R.; Murray, David L.; Hendrickson, Christopher L.; Marshall, Alan G.

Journal of the American Society for Mass Spectrometry (2017), 28 (5), 827-838CODEN: JAMSEF; ISSN:1044-0305. (Springer)

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clin. safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy anal. procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample prepn. with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 T FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error ∼4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind anal. of five therapeutic monoclonal antibodies at clin. relevant concns. in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resoln., mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS anal. of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and const. regions have been achieved for mAbs in a human serum background.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjsF2qtbc%253D&md5=c09c7faa3fbcd24bc7d40dec67de67f5

258

Tsybin, Y. O. ; Fornelli, L. ; Stoermer, C. ; Luebeck, M. ; Parra, J. ; Nallet, S. ; Wurm, F. M. ; Hartmer, R. Anal. Chem. 2011 , 83 , 8919 – 8927 DOI: 10.1021/ac201293m

[ACS Full Text ], [CAS], Google Scholar

258

Structural Analysis of Intact Monoclonal Antibodies by Electron Transfer Dissociation Mass Spectrometry

Tsybin, Yury O.; Fornelli, Luca; Stoermer, Carsten; Luebeck, Markus; Parra, Julien; Nallet, Sophie; Wurm, Florian M.; Hartmer, Ralf

Analytical Chemistry (Washington, DC, United States) (2011), 83 (23), 8919-8927CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Improving qual. and quant. characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissocn. (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, the authors describe the performance of ETD-based top-down mass spectrometry for structural anal. of intact 150 kDa monoclonal antibodies, Igs G (IgGs). Simultaneous mass anal. of intact IgGs as well as a complex mixt. of ETD product ions at sufficiently high resoln. and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resoln. ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissocn. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the expt. sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.

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259

Fornelli, L. ; Damoc, E. ; Thomas, P. M. ; Kelleher, N. L. ; Aizikov, K. ; Denisov, E. ; Makarov, A. ; Tsybin, Y. O. Mol. Cell. Proteomics 2012 , 11 , 1758 – 1767 DOI: 10.1074/mcp.M112.019620

[Crossref], [PubMed], [CAS], Google Scholar

259

Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation Orbitrap FTMS

Fornelli, Luca; Damoc, Eugen; Thomas, Paul M.; Kelleher, Neil L.; Aizikov, Konstantin; Denisov, Eduard; Makarov, Alexander; Tsybin, Yury O.

Molecular & Cellular Proteomics (2012), 11 (12), 1758-1767, 10 pp.CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure-function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact IgG (IgG), which includes a variable no. of disulfide bridges, its extensive fragmentation and subsequent sequence detn. by means of tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissocn. (ETD), implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS), to analyze a com. recombinant IgG in a liq. chromatog. (LC)-tandem mass spectrometry (MS/MS) top-down expt. The lack of sensitivity typically obsd. during the top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS expts. before performing Fourier transform signal processing. The results demonstrate that an improved signal-to-noise ratio, along with the higher resoln. and mass accuracy provided by Orbitrap FTMS (relative to previous applications of top-down ETD-based proteomics on IgG), is essential for comprehensive anal. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost 2-fold increase in IgG sequence coverage relative to prior ETD-based anal. of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodol. to other classes of large proteins and biomols.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvV2jsb%252FF&md5=5fcfe020f01975c0532f79b7f27813fc

260

Pan, J. ; Zhang, S. ; Chou, A. ; Hardie, D. B. ; Borchers, C. H. Anal. Chem. 2015 , 87 , 5884 – 5890 DOI: 10.1021/ac504809r

[ACS Full Text ], [CAS], Google Scholar

260

Fast Comparative Structural Characterization of Intact Therapeutic Antibodies Using Hydrogen-Deuterium Exchange and Electron Transfer Dissociation

Pan, Jingxi; Zhang, Suping; Chou, Albert; Hardie, Darryl B.; Borchers, Christoph H.

Analytical Chemistry (Washington, DC, United States) (2015), 87 (12), 5884-5890CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Higher-order structural characterization plays an important role in many stages of therapeutic antibody prodn. Herein, the authors report a new top-down mass spectrometry approach for characterizing the higher-order structure of intact antibodies, by combining hydrogen/deuterium exchange (HDX), subzero temp. chromatog., and electron transfer dissocn. on the Orbitrap mass spectrometer. Individual IgG domain-level deuteration information was obtained for 6 IgG domains on Herceptin (HER), which included the antigen binding sites. This is the first time that top-down HDX has been applied to an intact protein as large as 150 kDa, which has never been done before on any instrument. Ligand-binding induced structural differences in HER are located only on the variable region of the light chain. Global glycosylation profile of antibodies and HDX property of the glycoforms were also detd. by accurate intact mass measurements. Although the presence of disulfide bonds prevent the current approach from being able to obtain amino acid level structural information within the disulfide-linked regions, the advantages such as minimal sample manipulation, fast workflow, very low level of back exchange, and simple data anal., make it well-suited for fast comparative structural evaluation of intact antibodies.

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261

Fornelli, L. ; Ayoub, D. ; Aizikov, K. ; Liu, X. ; Damoc, E. ; Pevzner, P. A. ; Makarov, A. ; Beck, A. ; Tsybin, Y. O. J. Proteomics 2017 , 159 , 67 – 76 DOI: 10.1016/j.jprot.2017.02.013

[Crossref], [PubMed], [CAS], Google Scholar

261

Top-down analysis of immunoglobulin G isotypes 1 and 2 with electron transfer dissociation on a high-field Orbitrap mass spectrometer

Fornelli, Luca; Ayoub, Daniel; Aizikov, Konstantin; Liu, Xiaowen; Damoc, Eugen; Pevzner, Pavel A.; Makarov, Alexander; Beck, Alain; Tsybin, Yury O.

Journal of Proteomics (2017), 159 (), 67-76CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)

The increasing importance of Igs G (IgGs) as biotherapeutics calls for improved structural characterization methods designed for these large (∼ 150 kDa) macromols. Anal. workflows have to be rapid, robust, and require minimal sample prepn. In a previous work we showed the potential of Orbitrap Fourier transform mass spectrometry (FTMS) combined with electron transfer dissocn. (ETD) for the top-down investigation of an intact IgG1, resulting in ∼ 30% sequence coverage. Here, we describe a top-down anal. of two IgGs1 (adalimumab and trastuzumab) and one IgG2 (panitumumab) performed with ETD on a mass spectrometer equipped with a high-field Orbitrap mass analyzer. For the IgGs1, sequence coverage comparable to the previous results was achieved in a two-fold reduced no. of summed transients, which corresponds, taken together with the significantly increased spectra acquisition rate, to ∼ six-fold improvement in anal. time. Furthermore, we studied the influence of ion-ion interaction times on ETD product ions for IgGs1, and the differences in fragmentation behavior between IgGs1 and IgG2, which present structural differences. Overall, these results reinforce the hypothesis that gas phase dissocn. using both energy threshold-based and radical-driven ion activations is directed to specific regions of the polypeptide chains mostly by the location of disulfide bonds. Compared with our previous report, the results presented herein demonstrate the power of technol. advances of the next generation Orbitrap platform, including the use of a high-field compact (i.e., D20) Orbitrap mass analyzer, and a dedicated manipulation strategy for large protein ions (via their trapping in the HCD collision cell along with redn. of the pressure in the cell). Notably, these important developments became recently com. available in the top-end Orbitrap platforms under the name of "Protein Mode". Furthermore, we continued exploring the advantages offered by the summation (averaging) of transients (time-domain data) for improving the signal-to-noise ratio of top-down mass spectra. Finally, for the first time we report the application of the hybrid ion activation technique that combines electron transfer dissocn. and higher energy collisional dissocn., known as EThcD, on intact monoclonal antibodies. Under these specific instrumental parameters, EThcD produces a partially complementary fragmentation pattern compared to ETD, increasing the overall sequence coverage esp. at the protein termini.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXksFSjsb8%253D&md5=41acbc31d08926627830f6f1dd85ec36

262

Lössl, P. ; van de Waterbeemd, M. ; Heck, A. J. EMBO J. 2016 , 35 , 2634 – 2657 DOI: 10.15252/embj.201694818

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262

The diverse and expanding role of mass spectrometry in structural and molecular biology

Lossl Philip; van de Waterbeemd Michiel; Heck Albert Jr; Lossl Philip; van de Waterbeemd Michiel; Heck Albert Jr

The EMBO journal (2016), 35 (24), 2634-2657 ISSN:.

The emergence of proteomics has led to major technological advances in mass spectrometry (MS). These advancements not only benefitted MS-based high-throughput proteomics but also increased the impact of mass spectrometry on the field of structural and molecular biology. Here, we review how state-of-the-art MS methods, including native MS, top-down protein sequencing, cross-linking-MS, and hydrogen-deuterium exchange-MS, nowadays enable the characterization of biomolecular structures, functions, and interactions. In particular, we focus on the role of mass spectrometry in integrated structural and molecular biology investigations of biological macromolecular complexes and cellular machineries, highlighting work on CRISPR-Cas systems and eukaryotic transcription complexes.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2snhtlKgsQ%253D%253D&md5=7169a241083c8594be96b643b1dc70b8

263

Liu, F. ; Rijkers, D. T. S. ; Post, H. ; Heck, A. J. R. Nat. Methods 2015 , 12 , 1179 – 1184 DOI: 10.1038/nmeth.3603

[Crossref], [PubMed], [CAS], Google Scholar

263

Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry

Liu, Fan; Rijkers, Dirk T. S.; Post, Harm; Heck, Albert J. R.

Nature Methods (2015), 12 (12), 1179-1184CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

We describe an integrated workflow that robustly identifies cross-links from endogenous protein complexes in human cellular lysates. Our approach is based on the application of mass spectrometry (MS)-cleavable cross-linkers, sequential collision-induced dissocn. (CID)-tandem MS (MS/MS) and electron-transfer dissocn. (ETD)-MS/MS acquisitions, and a dedicated search engine, XlinkX, which allows rapid cross-link identification against a complete human proteome database. This approach allowed us to detect 2,179 unique cross-links (1,665 intraprotein cross-links at a 5% false discovery rate (FDR) and 514 interprotein cross-links at 1% FDR) in HeLa cell lysates. We validated the confidence of our crosslinking results by using a target-decoy strategy and mapping the obsd. cross-link distances onto existing high-resoln. structures. Our data provided new structural information about many protein assemblies and captured dynamic interactions of the ribosome in contact with different elongation factors.

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264

Liu, F. ; Lössl, P. ; Scheltema, R. ; Viner, R. ; Heck, A. J. R. Nat. Commun. 2017 , 8 , 15473 DOI: 10.1038/ncomms15473

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264

Optimized fragmentation schemes and data analysis strategies for proteome-wide cross-link identification

Liu, Fan; Loessl, Philip; Scheltema, Richard; Viner, Rosa; Heck, Albert J. R.

Nature Communications (2017), 8 (), 15473CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)

We describe optimized fragmentation schemes and data anal. strategies substantially enhancing the depth and accuracy in identifying protein cross-links using non-restricted whole proteome databases. These include a novel hybrid data acquisition strategy to sequence cross-links at both MS2 and MS3 level and a new algorithmic design XlinkX v2.0 for data anal. As proof-of-concept we investigated proteome-wide protein interactions in E. coli and HeLa cell lysates, resp., identifying 1,158 and 3,301 unique cross-links at ~ 1% false discovery rate. These protein interaction repositories provide meaningful structural information on many endogenous macromol. assemblies, as we showcase on several protein complexes involved in translation, protein folding and carbohydrate metab.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXot1Wlurw%253D&md5=ca5c671ac595773e1aecefa279536e80

265

Giese, S. H. ; Belsom, A. ; Rappsilber, J. Anal. Chem. 2016 , 88 , 8239 – 8247 DOI: 10.1021/acs.analchem.6b02082

266

Kolbowski, L. ; Mendes, M. L. ; Rappsilber, J. Anal. Chem. 2017 , 89 , 5311 – 5318 DOI: 10.1021/acs.analchem.6b04935

267

Sohn, C. H. ; Agnew, H. D. ; Lee, J. E. ; Sweredoski, M. J. ; Graham, R. L. J. ; Smith, G. T. ; Hess, S. ; Czerwieniec, G. ; Loo, J. A. ; Heath, J. R. ; Deshaies, R. J. ; Beauchamp, J. L. Anal. Chem. 2012 , 84 , 2662 – 2669 DOI: 10.1021/ac202637n

268

Koolen, H. H. F. ; Gomes, A. F. ; Schwab, N. V. ; Eberlin, M. N. ; Gozzo, F. C. J. Am. Soc. Mass Spectrom. 2014 , 25 , 1181 – 1191 DOI: 10.1007/s13361-014-0900-5

269

Lermyte, F. ; Sobott, F. Proteomics 2015 , 15 , 2813 – 2822 DOI: 10.1002/pmic.201400516

270

Lermyte, F. ; Konijnenberg, A. ; Williams, J. P. ; Brown, J. M. ; Valkenborg, D. ; Sobott, F. J. Am. Soc. Mass Spectrom. 2014 , 25 , 343 – 350 DOI: 10.1007/s13361-013-0798-3

271

Lermyte, F. ; Łącki, M. K. ; Valkenborg, D. ; Gambin, A. ; Sobott, F. J. Am. Soc. Mass Spectrom. 2017 , 28 , 69 – 76 DOI: 10.1007/s13361-016-1444-7

272

Zhang, Z. ; Browne, S. J. ; Vachet, R. W. J. Am. Soc. Mass Spectrom. 2014 , 25 , 604 – 613 DOI: 10.1007/s13361-013-0821-8

273

Zhang, Z. ; Vachet, R. W. Gas-phase protein salt bridge stabilities from collisional activation and electron transfer dissociation Int. J. Mass Spectrom. 2017 , 420 , 51 – 56 DOI: 10.1016/j.ijms.2016.09.010

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273

Gas-phase protein salt bridge stabilities from collisional activation and electron transfer dissociation

Zhang, Zhe; Vachet, Richard W.

International Journal of Mass Spectrometry (2017), 420 (), 51-56CODEN: IMSPF8; ISSN:1387-3806. (Elsevier B.V.)

The gas phase structures of several proteins have been studied by electron transfer dissocn. (ETD) with and without prior collisional heating after electrospraying these proteins from native-like solns. into a quadrupole ion trap mass spectrometer. Without prior collisional heating, we find that ETD fragmentation is mostly limited to regions of the protein that are not spanned by the salt bridges known to form in soln. When protein ions are collisionally heated before ETD, new product ions are obsd., and in almost all cases, these new ions arise from protein regions that are spanned by the salt bridges. Together these results confirm the existence of salt bridges in protein ions and demonstrate that a sufficient amt. energy is required to disrupt these salt bridges in the gas phase. More interestingly, we also show that different salt bridges require different collisional activation voltages to be disrupted, suggesting that they have variable stabilities in the gas phase. These stabilities appear to be influenced by the gas-phase basicities of the involved residues and the presence of nearby charged residues. We also find that higher collisional activation voltages are needed to enable the formation of new product from sites spanned by multiple salt bridges.

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274

Compton, P. D. ; Fornelli, L. ; Kelleher, N. L. ; Skinner, O. S. Int. J. Mass Spectrom. 2015 , 390 , 132 – 136 DOI: 10.1016/j.ijms.2015.08.021

275

Muneeruddin, K. ; Nazzaro, M. ; Kaltashov, I. A. Anal. Chem. 2015 , 87 , 10138 – 10145 DOI: 10.1021/acs.analchem.5b02982

[ACS Full Text ], [CAS], Google Scholar

275

Characterization of Intact Protein Conjugates and Biopharmaceuticals Using Ion-Exchange Chromatography with Online Detection by Native Electrospray Ionization Mass Spectrometry and Top-Down Tandem Mass Spectrometry

Muneeruddin, Khaja; Nazzaro, Mark; Kaltashov, Igor A.

Analytical Chemistry (Washington, DC, United States) (2015), 87 (19), 10138-10145CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Characterization of biopharmaceutical products is a challenging task, which needs to be carried out at several different levels (including both primary structure and conformation). An addnl. difficulty frequently arises due to the structural heterogeneity inherent to many protein-based therapeutics (e.g., extensive glycosylation or "designer" modifications such as chem. conjugation) or introduced postprodn. as a result of stress (e.g., oxidn. and deamidation). A combination of ion-exchange chromatog. (IXC) with online detection by native electrospray ionization mass spectrometry (ESI MS) allows characterization of complex and heterogeneous therapeutic proteins and protein conjugates to be accomplished at a variety of levels without compromising their conformational integrity. The IXC/ESI MS measurements allow protein conjugates to be profiled by analyzing conjugation stoichiometry and the presence of multiple positional isomers, as well as to establish the effect of chem. modifications on the conformational integrity of each species. While mass profiling alone is not sufficient for identification of nonenzymic post-translational modifications (PTMs) that result in a very small mass change of the eluting species (e.g., deamidation), this task can be completed using online top-down structural anal., as demonstrated using stressed interferon-β as an example. The wealth of information that can be provided by IXC/native ESI MS and tandem mass spectrometry (MS/MS) on protein-based therapeutics will undoubtedly make it a very valuable addn. to the exptl. toolbox of biopharmaceutical anal.

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276

Cassou, C. A. ; Sterling, H. J. ; Susa, A. C. ; Williams, E. R. Anal. Chem. 2013 , 85 , 138 – 146 DOI: 10.1021/ac302256d

[ACS Full Text ], [CAS], Google Scholar

276

Electrothermal Supercharging in Mass Spectrometry and Tandem Mass Spectrometry of Native Proteins

Cassou, Catherine A.; Sterling, Harry J.; Susa, Anna C.; Williams, Evan R.

Analytical Chemistry (Washington, DC, United States) (2013), 85 (1), 138-146CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Electrothermal supercharging of protein ions formed by electrospray ionization from buffered aq. solns. results in significant increases to both the max. and av. charge states compared to native mass spectrometry in which ions are formed from the same solns. but with lower spray potentials. For eight of the nine proteins studied, the max. charge states of protonated ions formed from native solns. with electrothermal supercharging is greater than those obtained from conventional denaturing solns. consisting of water/methanol/acid, although the av. charging is slightly lower owing to contributions of small populations of more folded low charge-state structures. Under these conditions, electrothermal supercharging is slightly less effective for anions than for cations. Equivalent sequence coverage (80%) was obtained with electron transfer dissocn. of the same high charge-state ion of cytochrome c formed by electrothermal supercharging from native solns. and from denaturing solns. Electrothermal supercharging should be advantageous for combining structural studies of proteins in native environments with mass spectrometers that have limited high m/z capabilities and for significantly improving tandem mass spectrometry performance for protein ions formed from solns. in which the mols. have native structures and activities.

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277

Xie, B. ; Sharp, J. S. J. Am. Soc. Mass Spectrom. 2016 , 27 , 1322 – 1327 DOI: 10.1007/s13361-016-1403-3

[Crossref], [PubMed], [CAS], Google Scholar

277

Relative Quantification of Sites of Peptide and Protein Modification Using Size Exclusion Chromatography Coupled with Electron Transfer Dissociation

Xie, Boer; Sharp, Joshua S.

Journal of the American Society for Mass Spectrometry (2016), 27 (8), 1322-1327CODEN: JAMSEF; ISSN:1044-0305. (Springer)

One difficult problem in the anal. of peptide modifications is quantifying isomeric modifications that differ by the position of the amino acid modified. HPLC sepn. using C18 reverse phase chromatog. coupled with electron transfer dissocn. (ETD) in tandem mass spectrometry has recently been shown to be able to relatively quantify how much of a given modification occurs at each amino acid position for isomeric mixts.; however, the resoln. of reverse phase chromatog. greatly complicates quantification of isomeric modifications by ETD because of the chromatog. sepn. of peptides with identical modifications at different sequence positions. Using peptide oxidn. as a model system, we investigated the use of size exclusion chromatog. coupled with ETD fragmentation to sep. peptide sequences. This approach allows for the benefits of chromatog. sepn. of peptide sequences while ensuring co-elution of modification isomers for accurate relative quantification of modifications using std. data-dependent acquisitions. Using this method, the relative amt. of modification at each amino acid can be accurately measured from single ETD MS/MS spectra in a std. data-dependent acquisition expt.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtVKhsrY%253D&md5=fbbb1a1763e033f1c0aaa8c39ada5d97

278

Zehl, M. ; Rand, K. D. ; Jensen, O. N. ; Jørgensen, T. J. D. J. Am. Chem. Soc. 2008 , 130 , 17453 – 17459 DOI: 10.1021/ja805573h

279

Abzalimov, R. R. ; Kaplan, D. A. ; Easterling, M. L. ; Kaltashov, I. A. J. Am. Soc. Mass Spectrom. 2009 , 20 , 1514 – 1517 DOI: 10.1016/j.jasms.2009.04.006

280

Landgraf, R. R. ; Chalmers, M. J. ; Griffin, P. R. J. Am. Soc. Mass Spectrom. 2012 , 23 , 301 – 309 DOI: 10.1007/s13361-011-0298-2

[Crossref], [PubMed], [CAS], Google Scholar

280

Automated hydrogen/deuterium exchange electron transfer dissociation high resolution mass spectrometry measured at single-amide resolution

Landgraf, Rachelle R.; Chalmers, Michael J.; Griffin, Patrick R.

Journal of the American Society for Mass Spectrometry (2012), 23 (2), 301-309CODEN: JAMSEF; ISSN:1044-0305. (Springer)

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a well established method for the measurement of soln.-phase deuterium incorporation into proteins, which can provide insight into protein conformational mobility. However, most HDX measurements are constrained to regions of the protein where pepsin proteolysis allows detection at peptide resoln. Recently, single-amide resoln. deuterium incorporation has been achieved by limiting gas-phase scrambling in the mass spectrometer. This was accomplished by employing a combination of soft ionization and desolvation conditions coupled with the radical-driven fragmentation technique electron transfer dissocn. (ETD). Here, a hybrid LTQ-Orbitrap XL is systematically evaluated for its utility in providing single-amide deuterium incorporation for differential HDX anal. of a nuclear receptor upon binding small mol. ligands. The authors are able to show that instrumental parameters can be optimized to minimize scrambling and can be incorporated into an established and fully automated HDX platform making differential single-amide HDX possible for bottom-up anal. of complex systems. The authors have applied this system to det. differential single amide resoln. HDX data for the peroxisome proliferator activated receptor bound with two ligands of interest.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XltVaiu7w%253D&md5=a525b532fbe74b28e6d66f5c28a5d131

281

Hamuro, Y. J. Am. Soc. Mass Spectrom. 2017 , 28 , 971 – 977 DOI: 10.1007/s13361-017-1612-4

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281

Regio-Selective Intramolecular Hydrogen/Deuterium Exchange in Gas-Phase Electron Transfer Dissociation

Hamuro, Yoshitomo

Journal of the American Society for Mass Spectrometry (2017), 28 (5), 971-977CODEN: JAMSEF; ISSN:1044-0305. (Springer)

Protein backbone amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) typically utilizes enzymic digestion after the exchange reaction and before MS anal. to improve data resoln. Gas-phase fragmentation of a peptic fragment prior to MS anal. is a promising technique to further increase the resoln. The biggest tech. challenge for this method is elimination of intramol. hydrogen/deuterium exchange (scrambling) in the gas phase. The scrambling obscures the location of deuterium. Jorgensen's group pioneered a method to minimize the scrambling in gas-phase electron capture/transfer dissocn. Despite active investigation, the mechanism of hydrogen scrambling is not well-understood. The difficulty stems from the fact that the degree of hydrogen scrambling depends on instruments, various parameters of mass anal., and peptide analyzed. In most hydrogen scrambling investigations, the hydrogen scrambling is measured by the percentage of scrambling in a whole mol. This paper demonstrates that the degree of intramol. hydrogen/deuterium exchange depends on the nature of exchangeable hydrogen sites. The deuterium on Tyr amide of neurotensin (9-13), Arg-Pro-Tyr-Ile-Leu, migrated significantly faster than that on Ile or Leu amides, indicating the loss of deuterium from the original sites is not mere randomization of hydrogen and deuterium but more site-specific phenomena. This more precise approach may help understand the mechanism of intramol. hydrogen exchange and provide higher confidence for the parameter optimization to eliminate intramol. hydrogen/deuterium exchange during gas-phase fragmentation.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXisF2mtLw%253D&md5=b6a3f6822f8db2396e29d5ec0a4015c1

282

Masson, G. R. ; Maslen, S. L. ; Williams, R. L. Biochem. J. 2017 , 474 , 1867 – 1877 DOI: 10.1042/BCJ20170127

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282

Analysis of phosphoinositide 3-kinase inhibitors by bottom-up electron-transfer dissociation hydrogen/deuterium exchange mass spectrometry

Masson, Glenn R.; Maslen, Sarah L.; Williams, Roger L.

Biochemical Journal (2017), 474 (11), 1867-1877CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)

Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resoln. afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissocn. to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS anal. is able to discern a conserved mechanism of inhibition common to a range of inhibitors. Owing to the relatively minor amts. of protein required, this technique may be utilized in pharmaceutical development for screening potential therapeutics.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXptFemsLk%253D&md5=948f40c1709b9d18010a6c645edd64f9

283

Seger, S. T. ; Breinholt, J. ; Faber, J. H. ; Andersen, M. D. ; Wiberg, C. ; Schjødt, C. B. ; Rand, K. D. Anal. Chem. 2015 , 87 , 5973 – 5980 DOI: 10.1021/ac504782v

[ACS Full Text ], [CAS], Google Scholar

283

Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen-Deuterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation

Seger, Signe T.; Breinholt, Jens; Faber, Johan H.; Andersen, Mette D.; Wiberg, Charlotte; Schjoedt, Christine B.; Rand, Kasper D.

Analytical Chemistry (Washington, DC, United States) (2015), 87 (12), 5973-5980CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helixes 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the impact of the new disulfide bond on the conformational dynamics of this new hGH variant. Compared to wild type hGH, the variant exhibits reduced loop dynamics, indicating a stabilizing effect of the introduced disulfide bond. Furthermore, the disulfide bond exhibits longer ranging effects, stabilizing a short α-helix quite distant from the mutation sites, but also rendering a part of the α-helical hGH core slightly more dynamic. In the regions where the hGH variant exhibits a different deuterium uptake than the wild type protein, electron transfer dissocn. (ETD) fragmentation has been used to pinpoint the residues responsible for the obsd. differences (HDX-ETD). Finally, by use of surface plasmon resonance (SPR) measurements, we show that the new disulfide bond does not compromise receptor affinity. Our work highlight the anal. potential of HDX-ETD combined with functional assays to guide protein engineering.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXosVCht78%253D&md5=a9d8ede319b2bb172d5341a6352cf626

284

Song, H. ; Olsen, O. H. ; Persson, E. ; Rand, K. D. J. Biol. Chem. 2014 , 289 , 35388 – 35396 DOI: 10.1074/jbc.M114.614297

[Crossref], [PubMed], [CAS], Google Scholar

284

Sites Involved in Intra- and Interdomain Allostery Associated with the Activation of Factor VIIa Pinpointed by Hydrogen-Deuterium Exchange and Electron Transfer Dissociation Mass Spectrometry

Song, Hongjian; Olsen, Ole H.; Persson, Egon; Rand, Kasper D.

Journal of Biological Chemistry (2014), 289 (51), 35388-35396CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)

Factor VIIa (FVIIa) is a trypsin-like protease that plays an important role in initiating blood coagulation. Very limited structural information is available for the free, inactive form of FVIIa that circulates in the blood prior to vascular injury and the mol. details of its activity enhancement remain elusive. Here we have applied hydrogen/deuterium exchange mass spectrometry coupled to electron transfer dissocn. to pinpoint individual residues in the heavy chain of FVIIa whose conformation and/or local interaction pattern changes when the enzyme transitions to the active form, as induced either by its cofactor tissue factor or a covalent active site inhibitor. Identified regulatory residues are situated at key sites across one continuous surface of the protease domain spanning the TF-binding helix across the activation pocket to the calcium binding site and are embedded in elements of secondary structure and at the base of flexible loops. Thus these residues are optimally positioned to mediate crosstalk between functional sites in FVIIa, particularly the cofactor binding site and the active site. Our results unambiguously show that the conformational allosteric activation signal extends to the EGF1 domain in the light chain of FVIIa, underscoring a remarkable intra- and interdomain allosteric regulation of this trypsin-like protease.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitFCrtrnO&md5=3c573ffece78a9a54d2b3cc45353ac12

285

Iacob, R. E. ; Chen, G. ; Ahn, J. ; Houel, S. ; Wei, H. ; Mo, J. ; Tao, L. ; Cohen, D. ; Xie, D. ; Lin, Z. ; Morin, P. E. ; Doyle, M. L. ; Tymiak, A. A. ; Engen, J. R. J. Am. Soc. Mass Spectrom. 2014 , 25 , 2093 – 2102 DOI: 10.1007/s13361-014-0973-1

286

Huang, R. Y.-C. ; Garai, K. ; Frieden, C. ; Gross, M. L. Biochemistry 2011 , 50 , 9273 – 9282 DOI: 10.1021/bi2010027

[ACS Full Text ], [CAS], Google Scholar

286

Hydrogen/Deuterium Exchange and Electron-Transfer Dissociation Mass Spectrometry Determine the Interface and Dynamics of Apolipoprotein E Oligomerization

Huang, Richard Y.-C.; Garai, Kanchan; Frieden, Carl; Gross, Michael L.

Biochemistry (2011), 50 (43), 9273-9282CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

Apolipoprotein E, a 34 kDa protein, plays a key role in triglyceride and cholesterol metab. Of the three common isoforms (ApoE2, -3, and -4), only ApoE4 is a risk factor for Alzheimer's disease. All three isoforms of wild-type ApoE self-assoc. to form oligomers, a process that may have functional consequences. Although the C-terminal domain, residues 216-299, of ApoE is believed to mediate self-assocn., the specific residues involved in this process are not known. Here we report the use of hydrogen/deuterium exchange (H/DX) coupled with enzymic digestion to identify those regions in the sequence of full-length apoE involved in oligomerization. For this detn., we compared the results of H/DX of the wild-type proteins and those of monomeric forms obtained by modifying four residues in the C-terminal domain. The three wild-type and mutant isoforms show similar structures based on their similar H/DX kinetics and extents of exchange. Regions of the C-terminus (residues 230-270) of the ApoE isoforms show significant differences of deuterium uptake between oligomeric and monomeric forms, confirming that oligomerization occurs at these regions. To achieve single amino acid resoln., we examd. the extents of H/DX by using electron transfer dissocn. (ETD) fragmentation of peptides representing selected regions of both the monomeric and the oligomeric forms of ApoE4. From these expts., we could identify the specific residues involved in ApoE oligomerization. In addn., our results verify that ApoE4 is composed of a compact structure at its N-terminal domain. Regions of C-terminal domain, however, appear to lack defined structure.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1Kjtr7E&md5=05ff68258679326f480cf491d061abf8

287

Pan, J. ; Zhang, S. ; Borchers, C. H. J. Proteomics 2016 , 134 , 138 – 143 DOI: 10.1016/j.jprot.2015.12.002

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287

Protein species-specific characterization of conformational change induced by multisite phosphorylation

Pan, Jingxi; Zhang, Suping; Borchers, Christoph H.

Journal of Proteomics (2016), 134 (), 138-143CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)

Phosphorylation is a central mechanism for regulating the structure and function of proteins in the cell, but accurate characterization of a specific protein phospho-species is challenging due to the difficulty of sepg. it from other species, as well as the limitations of the traditional structural methods. By using selective top-down ETD, we were able to identify six specific phospho-species of calmodulin (CaM). Phosphorylation of CaM at four sites by CK2 was found to follow a sequential order, with Ser81 as the first, Thr79 as the second, and Ser101 or Thr117 as the third. By combining top-down ETD with hydrogen/deuterium exchange, the impact of phosphorylation on CaM's structure was elucidated in a species-specific manner. A negligible structural effect was obsd. for mono-phosphorylation at Ser81, or di-phosphorylation at Ser81-Thr79, or tri-phosphorylation at Ser81-Thr79-Ser101 or Ser81-Thr79-Thr117. However, it was found that a significant phosphorylation-induced conformational change in CaM was caused by simultaneous phosphorylation at Ser101 and Thr117. The dramatically increased deuterium incorporation for residues between 102 and 119 strongly suggests that the structure of this region has been greatly changed.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVSrsrzI&md5=29cede4c621cd458450c5a94db5cd337

288

Rand, K. D. ; Pringle, S. D. ; Morris, M. ; Engen, J. R. ; Brown, J. M. J. Am. Soc. Mass Spectrom. 2011 , 22 , 1784 – 1793 DOI: 10.1007/s13361-011-0196-7

289

Khakinejad, M. ; Ghassabi Kondalaji, S. ; Tafreshian, A. ; Valentine, S. J. J. Am. Soc. Mass Spectrom. 2017 , 28 , 960 – 970 DOI: 10.1007/s13361-017-1641-z

290

Mistarz, U. H. ; Brown, J. M. ; Haselmann, K. F. ; Rand, K. D. Anal. Chem. 2014 , 86 , 11868 – 11876 DOI: 10.1021/ac5035456

291

Xie, B. ; Sood, A. ; Woods, R. J. ; Sharp, J. S. Sci. Rep. 2017 , 7 , 4552 DOI: 10.1038/s41598-017-04689-3

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291

Quantitative Protein Topography Measurements by High Resolution Hydroxyl Radical Protein Footprinting Enable Accurate Molecular Model Selection

Xie Boer; Sood Amika; Woods Robert J; Sharp Joshua S

Scientific reports (2017), 7 (1), 4552 ISSN:.

We report an integrated workflow that allows mass spectrometry-based high-resolution hydroxyl radical protein footprinting (HR-HRPF) measurements to accurately measure the absolute average solvent accessible surface area (<SASA>) of amino acid side chains. This approach is based on application of multi-point HR-HRPF, electron-transfer dissociation (ETD) tandem MS (MS/MS) acquisition, measurement of effective radical doses by radical dosimetry, and proper normalization of the inherent reactivity of the amino acids. The accuracy of the resulting <SASA> measurements was tested by using well-characterized protein models. Moreover, we demonstrated the ability to use <SASA> measurements from HR-HRPF to differentiate molecular models of high accuracy (<3 ÅA backbone RMSD) from models of lower accuracy (>4 ÅA backbone RMSD). The ability of <SASA> data from HR-HRPF to differentiate molecular model quality was found to be comparable to that of <SASA> data obtained from X-ray crystal structures, indicating the accuracy and utility of HR-HRPF for evaluating the accuracy of computational models.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cjkt1Clsg%253D%253D&md5=4ee24603a048e9303017e98e19b97a82

292

Vasicek, L. ; O'Brien, J. P. ; Browning, K. S. ; Tao, Z. ; Liu, H.-W. ; Brodbelt, J. S. Mol. Cell. Proteomics 2012 , 11 , O111.015826 DOI: 10.1074/mcp.O111.015826

293

Budelier, M. M. ; Cheng, W. W. L. ; Bergdoll, L. ; Chen, Z.-W. ; Abramson, J. ; Krishnan, K. ; Qian, M. ; Covey, D. F. ; Janetka, J. W. ; Evers, A. S. Anal. Chem. 2017 , 89 , 2636 – 2644 DOI: 10.1021/acs.analchem.6b05003

294

Prentice, B. M. ; McLuckey, S. A. Chem. Commun. 2013 , 49 , 947 – 965 DOI: 10.1039/C2CC36577D

295

Gilbert, J. D. ; Fisher, C. M. ; Bu, J. ; Prentice, B. M. ; Redwine, J. G. ; McLuckey, S. A. J. Mass Spectrom. 2015 , 50 , 418 – 426 DOI: 10.1002/jms.3548

296

Hurtado, P. P. ; O'Connor, P. B. Mass Spectrom. Rev. 2012 , 31 , 609 – 625 DOI: 10.1002/mas.20357

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296

Differentiation of isomeric amino acid residues in proteins and peptides using mass spectrometry

Hurtado, Pilar Perez; O'Connor, Peter B.

Mass Spectrometry Reviews (2012), 31 (6), 609-625CODEN: MSRVD3; ISSN:0277-7037. (John Wiley & Sons, Inc.)

A review. Characterization and differentiation of isomers in biol. macromols. using mass spectrometry is one of the most significant challenges facing scientists in the field. The capability of high-resoln. MS instruments along with the development of new fragmentation methods now provides the ability to indirectly differentiate between some isomers. This ability has enabled mass spectrometry to evolve into a multidisciplinary technique incorporating areas such as pharmaceutical research, proteomics, polymer science, medicine, environmental chem., and recently archeol. This article aims to review recent developments in mass spectrometry methodologies in the identification of structural and spatial isomers in biol. macromols., such as aspartic acid and isoaspartic acid (Asp/IsoAsp), leucine and isoleucine (Leu/Ile), glutamic acid and γ-glutamic acid, and D/L enantiomers. © 2012 Wiley Periodicals, Inc. Mass Spec Rev 31:609-625, 2012.

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297

Lebedev, A. T. ; Damoc, E. ; Makarov, A. A. ; Samgina, T. Y. Anal. Chem. 2014 , 86 , 7017 – 7022 DOI: 10.1021/ac501200h

298

Xiao, Y. ; Vecchi, M. M. ; Wen, D. Anal. Chem. 2016 , 88 , 10757 – 10766 DOI: 10.1021/acs.analchem.6b03409

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298

Distinguishing between Leucine and Isoleucine by Integrated LC-MS Analysis Using an Orbitrap Fusion Mass Spectrometer

Xiao, Yongsheng; Vecchi, Malgorzata M.; Wen, Dingyi

Analytical Chemistry (Washington, DC, United States) (2016), 88 (21), 10757-10766CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Despite the great success of mass spectrometry (MS) for de novo protein sequencing, Leu and Ile have been generally considered to be indistinguishable by MS because their mol. masses are exactly the same. Positioning of incorrect Leu/Ile residues in variable domains, esp. in CDRs (complementarity detg. regions) of an antibody, may result in substantial loss of antigen binding affinity and specificity of the antibody. Here, the authors describe an integrated LC-MS based strategy, encompassing a combination of HCD (high-energy collisional dissocn.) multistage mass spectrometric anal. (HCD-MSn) and ETD (electron transfer dissocn.)-HCD MS3 anal. using an Orbitrap Fusion mass spectrometer, to reliably identify Leu and Ile residues in proteins and peptides. The merits and limitations of this Leu/Ile discrimination approach are evaluated. Using the new approach, along with proposed decision-making guidelines the authors unambiguously identified every Leu/Ile residue in peptides contg. up to five Leu/Ile residues and mol. masses up to 3000 Da. In addn., the authors have demonstrated, for the first time, that every Leu/Ile residue in the variable regions of a monoclonal antibody that could not be assigned by antibody germline sequence alignment could be correctly detd. using this approach. The results suggest that, by incorporating this approach into existing de novo antibody sequencing protocols, 100% of antibody amino acid sequences, including identity of Leu and Ile residues, can be accurately obtained solely by means of mass spectrometry. In principle, this integrated, online LC-MS approach for Leu/Ile assignment can be applied to de novo sequencing of any protein or peptide.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1WktbfP&md5=b525e9f63613ef86b3739fca099d9662

299

Zhokhov, S. S. ; Kovalyov, S. V. ; Samgina, T. Y. ; Lebedev, A. T. J. Am. Soc. Mass Spectrom. 2017 , 28 , 1600 – 1611 DOI: 10.1007/s13361-017-1674-3

300

Kovalyov, S. V. ; Zhokhov, S. S. ; Onoprienko, L. V. ; Vaskovsky, B. V. ; Lebedev, A. T. Eur. J. Mass Spectrom. 2017 , 23 , 376 – 384 DOI: 10.1177/1469066717730705

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300

Exploration of doubtful cases of leucine and isoleucine discrimination in mass spectrometric peptide sequencing by electron-transfer and higher-energy collision dissociation-based method

Kovalyov Sergey V; Zhokhov Sergey S; Lebedev Albert T; Onoprienko Ludmila V; Vaskovsky Boris V

European journal of mass spectrometry (Chichester, England) (2017), 23 (6), 376-384 ISSN:1469-0667.

Electron-transfer dissociation (ETD) and electron-transfer and higher-energy collision dissociation (EThcD) spectra of short tryptic peptides with leucine/isoleucine residues in neighboring positions demonstrate intensive w-ions. On the contrary, u-ions possess very low intensities (if present at all). Therefore radical site migration is negligible in the applied conditions while ETD (EThcD) spectra allow for the reliable discrimination of the isomeric residues in the sequencing process. The presence of a fragment ion 43.055 mass units lower than z2-ion of peptides with IK sequence at their C-termini was shown to be a result of alternative fragmentation starting from the loss of propylammonium ion from the doubly protonated peptide molecule and formation of an oxazole fragment ion.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M3lslCqtw%253D%253D&md5=b85a7c42eef0084c2ee541024f0c550a

301

Lyon, Y. A. ; Beran, G. ; Julian, R. R. J. Am. Soc. Mass Spectrom. 2017 , 28 , 1365 – 1373 DOI: 10.1007/s13361-017-1627-x

302

Ledvina, A. R. ; Coon, J. J. ; Tureček, F. Int. J. Mass Spectrom. 2015 , 377 , 44 – 53 DOI: 10.1016/j.ijms.2014.02.015

303

Nguyen, H. T. H. ; Shaffer, C. J. ; Ledvina, A. R. ; Coon, J. J. ; Tureček, F. Int. J. Mass Spectrom. 2015 , 378 , 20 – 30 DOI: 10.1016/j.ijms.2014.06.028

304

Nguyen, H. T. H. ; Shaffer, C. J. ; Tureček, F. J. Phys. Chem. B 2015 , 119 , 3948 – 3961 DOI: 10.1021/jp511717c

305

Nguyen, H. T. H. ; Tureček, F. J. Am. Soc. Mass Spectrom. 2017 , 28 , 1333 – 1344 DOI: 10.1007/s13361-016-1586-7

306

Shaffer, C. J. ; Marek, A. ; Pepin, R. ; Slovakova, K. ; Turecek, F. J. Mass Spectrom. 2015 , 50 , 470 – 475 DOI: 10.1002/jms.3551

307

Shaffer, C. J. ; Pepin, R. ; Tureček, F. J. Mass Spectrom. 2015 , 50 , 1438 – 1442 DOI: 10.1002/jms.3717

308

Pepin, R. ; Layton, E. D. ; Liu, Y. ; Afonso, C. ; Tureček, F. J. Am. Soc. Mass Spectrom. 2017 , 28 , 164 – 181 DOI: 10.1007/s13361-016-1512-z

309

McLuckey, S. A. ; Stephenson, J. L. Mass Spectrom. Rev. 1998 , 17 , 369 – 407 DOI: 10.1002/(SICI)1098-2787(1998)17:6<369::AID-MAS1>3.0.CO;2-J

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309

Ion/ion chemistry of high-mass multiply charged ions

McLuckey S A; Stephenson J L Jr

Mass spectrometry reviews (1998), 17 (6), 369-407 ISSN:0277-7037.

Electrospray ionization has enabled the establishment of a new area of ion chemistry research based on the study of the reactions of high-mass multiply charged ions with ions of opposite polarity. The multiple-charging phenomenon associated with electrospray makes possible the generation of multiply charged reactant ions that yield charged products as a result of partial neutralization due to ion/ion chemistry. The charged products can be readily studied with mass spectrometric methods, providing useful insights into reaction mechanisms. This review presents the research done in this area, all of which has been performed within the past decade. Ion/ion chemistry has been studied at near-atmospheric pressure in a reaction region that leads to the atmospheric/vacuum interface of a mass spectrometer, and within a quadrupole ion trap operated with a bath gas at a pressure of 1 mtorr. Proton transfer has been the most common reaction type for high-mass ions, but other forms of "charge transfer," such as electron transfer and fluoride transfer, have also been observed. For some ion/ion reactions, attachment of the two reactants has been observed. Multiply charged ion/ion reactions are fast, due to the long-range Coulombic attraction, and they are universal in that any pair of oppositely charged ions is expected to react due to the high exothermicity associated with mutual neutralization. The kinetics of reaction for multiply charged ions, derived from the same molecule with a given singly charged reactant ion, follow a charge-squared dependence, at least under normal quadrupole ion trap conditions. This dependence suggests that reaction rates are determined by the long-range Coulomb attraction, and that the ions react with constant efficiency as a function of charge state. In the case of proton transfer reactions from polypeptides to even-electron perfluorocarbon anions, no fragmentation of the polypeptide product ions has, as yet, been observed. Electron transfer from small oligonucleotide anions to rare gas cations, on the other hand, results in extensive fragmentation of the nucleic acid product ions. The extent of fragmentation decreases as the size of the oligonucleotide anions increases, reflecting a decrease in fragmentation rates associated with an increase in the number of internal degrees of freedom of the oligonucleotide. When ion-cooling rates become competitive with dissociation rates, the initially formed product ions are stabilized and fragmentation is avoided. Collisional cooling, therefore, likely plays an important role in the relative lack of dissociation observed thus far as a result of ion/ion reactions for most high-mass ions. The observed dependence of ion/ion reaction rates on the square of the ion charge, the universal nature of mutual neutralization, and the relative lack of fragmentation that arises from ion/ion reactions, makes ion/ion chemistry a particularly useful means for manipulating charge states. This review emphasizes applications that take advantage of the unique characteristics of ion/ion proton transfer chemistry for manipulating charge states. These applications include mixture analysis by electrospray, precursor ion charge state manipulation for tandem mass spectrometry studies, and simplified interpretation of product ion spectra.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK1M3ovVKisw%253D%253D&md5=092af576e5bfbd3114c5b29e776b931b

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Reid, G. E. ; McLuckey, S. A. J. Mass Spectrom. 2002 , 37 , 663 – 675 DOI: 10.1002/jms.346

311

McLuckey, S. A. ; Huang, T.-Y. Anal. Chem. 2009 , 81 , 8669 – 8676 DOI: 10.1021/ac9014935

312

Drabik, A. ; Bodzon-Kulakowska, A. ; Suder, P. J. Mass Spectrom. 2012 , 47 , 1347 – 1352 DOI: 10.1002/jms.3086

313

Anderson, L. C. ; English, A. M. ; Wang, W.-H. ; Bai, D. L. ; Shabanowitz, J. ; Hunt, D. F. Int. J. Mass Spectrom. 2015 , 377 , 617 – 624 DOI: 10.1016/j.ijms.2014.06.023

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313

Protein derivatization and sequential ion/ion reactions to enhance sequence coverage produced by electron transfer dissociation mass spectrometry

Anderson, Lissa C.; English, A. Michelle; Wang, Wei-Han; Bai, Dina L.; Shabanowitz, Jeffrey; Hunt, Donald F.

International Journal of Mass Spectrometry (2015), 377 (), 617-624CODEN: IMSPF8; ISSN:1387-3806. (Elsevier B.V.)

Previously, we described implementation of a front-end ETD (electron transfer dissocn.) source for an Orbitrap instrument [1]. This source facilitates multiple fills of the C-trap with product ions from ETD of intact proteins prior to mass anal. The result is a dramatic enhancement of the obsd. ion current without the need for time consuming averaging of data from multiple mass measurements. Here we show that ion-ion proton transfer (IIPT) reactions can be used to simplify ETD spectra and to disperse fragment ions over the entire mass range in a controlled manner. We also show that protein derivatization can be employed to selectively enhance the sequence information obsd. at the N- and C-termini of a protein.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht1Cht7vM&md5=7aeade9cb25c0d26e756194761dee6ed

314

Chrisman, P. A. ; Pitteri, S. J. ; McLuckey, S. A. Anal. Chem. 2006 , 78 , 310 – 316 DOI: 10.1021/ac0515778

315

Holden, D. D. ; McGee, W. M. ; Brodbelt, J. S. Anal. Chem. 2016 , 88 , 1008 – 1016 DOI: 10.1021/acs.analchem.5b03911

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315

Integration of Ultraviolet Photodissociation with Proton Transfer Reactions and Ion Parking for Analysis of Intact Proteins

Holden, Dustin D.; McGee, William M.; Brodbelt, Jennifer S.

Analytical Chemistry (Washington, DC, United States) (2016), 88 (1), 1008-1016CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

We report the implementation of proton transfer reactions (PTR) and ion parking on an Orbitrap mass spectrometer. PTR/ion parking allows charge states of proteins to be focused into a single lower charge state via sequential deprotonation reactions with a proton scavenging reagent, in this case, a nitrogen-contg. adduct of fluoranthene. Using PTR and ion parking, we evaluate the charge state dependence of fragmentation of ubiquitin (8.6 kDa), myoglobin (17 kDa), and carbonic anhydrase (29 kDa) upon higher energy collisional dissocn. (HCD) or UV photodissocn. (UVPD). UVPD exhibited less charge state dependence, thus yielding more uniform distributions of cleavages along the protein backbone and consequently higher sequence coverage than HCD. HCD resulted in esp. prominent cleavages C-terminal to amino acids contg. acidic side-chains and N-terminal to proline residues; UVPD did not exhibit preferential cleavage adjacent to acidic residues but did show enhancement next to proline and phenylalanine.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFGns7%252FF&md5=65beb92ba774c1247733bc314d8473a2

316

Holden, D. D. ; Brodbelt, J. S. Anal. Chem. 2016 , 88 , 12354 – 12362 DOI: 10.1021/acs.analchem.6b03565

317

Wenger, C. D. ; Lee, M. V. ; Hebert, A. S. ; McAlister, G. C. ; Phanstiel, D. H. ; Westphall, M. S. ; Coon, J. J. Nat. Methods 2011 , 8 , 933 – 935 DOI: 10.1038/nmeth.1716

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317

Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging

Wenger, Craig D.; Lee, M. Violet; Hebert, Alexander S.; McAlister, Graeme C.; Phanstiel, Douglas H.; Westphall, Michael S.; Coon, Joshua J.

Nature Methods (2011), 8 (11), 933-935CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

We describe a mass spectrometry method, QuantMode, which improves accuracy of isobaric tag-based quantification by alleviating the pervasive problem of precursor interference, simultaneous isolation and fragmentation of impurities, through gas-phase purifn. QuantMode anal. of a yeast sample 'contaminated' with interfering human peptides showed substantially improved quant. accuracy compared to a std. scan, with a small loss of spectral identifications. This technique enables large-scale, multiplexed quant. proteomics using isobaric tagging.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1CrtrvJ&md5=64ded63680bef867d5e4690e9b58a5a5

318

Vincent, C. E. ; Rensvold, J. W. ; Westphall, M. S. ; Pagliarini, D. J. ; Coon, J. J. Anal. Chem. 2013 , 85 , 2079 – 2086 DOI: 10.1021/ac302156t

319

Laszlo, K. J. ; Bush, M. F. J. Am. Soc. Mass Spectrom. 2015 , 26 , 2152 – 2161 DOI: 10.1007/s13361-015-1245-4

320

Laszlo, K. J. ; Munger, E. B. ; Bush, M. F. J. Am. Chem. Soc. 2016 , 138 , 9581 – 9588 DOI: 10.1021/jacs.6b04282

321

Laszlo, K. J. ; Bush, M. F. Anal. Chem. 2017 , 89 , 7607 – 7614 DOI: 10.1021/acs.analchem.7b01474

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321

Interpreting the Collision Cross Sections of Native-like Protein Ions: Insights from Cation-to-Anion Proton-Transfer Reactions

Laszlo, Kenneth J.; Bush, Matthew F.

Analytical Chemistry (Washington, DC, United States) (2017), 89 (14), 7607-7614CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

The effects of charge state on structures of native-like cations of serum albumin, streptavidin, avidin, and alc. dehydrogenase were probed using cation-to-anion proton-transfer reactions (CAPTR), ion mobility, mass spectrometry, and complementary energy-dependent expts. The CAPTR products all have collision cross-section (Ω) values that are within 5.5% of the original precursor cations. The first CAPTR event for each precursor yields products that have smaller Ω values and frequently exhibit the greatest magnitude of change in Ω resulting from a single CAPTR event. To investigate how the structures of the precursors affect the structures of the products, ions were activated as a function of energy prior to CAPTR. In each case, the Ω values of the activated precursors increase with increasing energy, but the Ω values of the CAPTR products are smaller than the activated precursors. To investigate the stabilities of the CAPTR products, the products were activated immediately prior to ion mobility. These results show that addnl. structures with smaller or larger Ω values can be populated and that the structures and stabilities of these ions depend most strongly on the identity of the protein and the charge state of the product, rather than the charge state of the precursor or the no. of CAPTR events. Together, these results indicate that the excess charges initially present on native-like ions have a modest, but sometimes statistically significant, effect on their Ω values. Therefore, potential contributions from charge state should be considered when using exptl. Ω values to elucidate structures in soln.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVeku7bL&md5=49550a71ae0a11feae62d2977e9f6062

322

Laszlo, K. J. ; Buckner, J. H. ; Munger, E. B. ; Bush, M. F. J. Am. Soc. Mass Spectrom. 2017 , 28 , 1382 – 1391 DOI: 10.1007/s13361-017-1620-4

323

Jhingree, J. R. ; Beveridge, R. ; Dickinson, E. R. ; Williams, J. P. ; Brown, J. M. ; Bellina, B. ; Barran, P. E. Int. J. Mass Spectrom. 2017 , 413 , 43 – 51 DOI: 10.1016/j.ijms.2016.08.006

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323

Electron transfer with no dissociation ion mobility-mass spectrometry (ETnoD IM-MS). The effect of charge reduction on protein conformation

Jhingree, Jacquelyn R.; Beveridge, Rebecca; Dickinson, Eleanor R.; Williams, Jonathan P.; Brown, Jeffery M.; Bellina, Bruno; Barran, Perdita E.

International Journal of Mass Spectrometry (2017), 413 (), 43-51CODEN: IMSPF8; ISSN:1387-3806. (Elsevier B.V.)

A novel mass spectrometry approach is reported which investigate how ion-mol. charge redn. reactions between radical anions and protein cations modulate protein conformation. An electron transfer reagent (1,3-dicyanobenzene) transfers electrons to pos. charged proteins and there are no observable products of dissocn. ETnoD product ions are detected as charged-reduced species with the same mol. wt. as the precursor ion, and no significant evidence for proton transfer. We present collision cross section distributions of precursor and product ions before and after exposure to radical ions. Cytochrome c and myoglobin are examd. as exemplar systems under both aq. salt and denaturing conditions before and after exposure to radical anions. We consistently observe depletion of the more compact precursor ion conformers on exposure to the ETD reagent. Remarkably, by examg. the collision cross section distributions of the product ions it can be seen that the addn. of a single electron can cause a dramatic rearrangement in protein conformation for charge states that are highly populated when sprayed from salty aq. conditions. Furthermore, a given net charge on an exposed precursor and product ion favors a preferred collision cross section distribution, indicating that the distribution of charge on proteins in the gas phase dictate their conformation. An exception is reported for the low charge state of cytochrome c where compaction was seen in the radical formed post redn. compared to the electrospray generated ion under ETnoD optimized conditions. We propose a model that postulates how electron transfer to conformation stabilizing salt bridges may explain our observations.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXptFCjt7g%253D&md5=d6894977fc15cfe4a27d3e7e979e1188

324

Lermyte, F. ; Williams, J. P. ; Brown, J. M. ; Martin, E. M. ; Sobott, F. J. Am. Soc. Mass Spectrom. 2015 , 26 , 1068 – 1076 DOI: 10.1007/s13361-015-1124-z

325

Pilo, A. L. ; Zhao, F. ; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2017 , 28 , 991 – 1004 DOI: 10.1007/s13361-016-1554-2

326

Gilbert, J. D. ; Prentice, B. M. ; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2015 , 26 , 818 – 825 DOI: 10.1007/s13361-015-1077-2

327

Bu, J. ; Pilo, A. L. ; McLuckey, S. A. Int. J. Mass Spectrom. 2015 , 390 , 118 – 123 DOI: 10.1016/j.ijms.2015.05.010

328

Pilo, A. L. ; Peng, Z. ; McLuckey, S. A. J. Mass Spectrom. 2016 , 51 , 857 – 866 DOI: 10.1002/jms.3831

329

Peng, Z. ; Bu, J. ; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2017 , 28 , 1765 – 1774 DOI: 10.1007/s13361-017-1672-5

330

Pilo, A. L. ; Zhao, F. ; McLuckey, S. A. J. Proteome Res. 2016 , 15 , 3139 – 3146 DOI: 10.1021/acs.jproteome.6b00266

[ACS Full Text ], [CAS], Google Scholar

330

Selective Gas-Phase Oxidation and Localization of Alkylated Cysteine Residues in Polypeptide Ions via Ion/Ion Chemistry

Pilo, Alice L.; Zhao, Feifei; McLuckey, Scott A.

Journal of Proteome Research (2016), 15 (9), 3139-3146CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

The terminal thiol group in cysteine residues is susceptible to several post-translational modifications (PTMs), including prenylation, nitrosylation, palmitoylation, and the formation of disulfide bonds. Addnl., cysteine residues involved in disulfide bonds are commonly reduced and alkylated prior to mass spectrometric anal. Several of these cysteine modifications, specifically S-alkyl modifications, are susceptible to gas-phase oxidn. via selective ion/ion reactions with periodate anions. Multiply protonated peptides contg. modified cysteine residues undergo complex formation upon ion/ion reaction with periodate anions. Activation of the ion/ion complexes results in oxygen transfer from the reagent to the modified sulfur residue to create a sulfoxide functionality. Further activation of the sulfoxide deriv. yields abundant losses of the modification with the oxidized sulfur as a sulfenic acid (viz., XSOH) to generate a dehydroalanine residue. This loss immediately indicates the presence of an S-alkyl cysteine residue, and the mass of the loss can be used to easily deduce the type of modification. An addnl. step of activation can be used to localize the modification to a specific residue within the peptide. Selective cleavage to create c- and z-ions N-terminal to the dehydroalanine residue is often noted. As these types of ions are not typically obsd. upon collision-induced dissocn. (CID), they can be used to immediately indicate where in the peptide the PTM was originally located.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1Gmt73I&md5=218cf8b8bc262b1cb195e94588038968

331

Pilo, A. L. ; McLuckey, S. A. Anal. Chem. 2016 , 88 , 8972 – 8979 DOI: 10.1021/acs.analchem.6b01043

332

McGee, W. M. ; McLuckey, S. A. Proc. Natl. Acad. Sci. U. S. A. 2014 , 111 , 1288 – 1292 DOI: 10.1073/pnas.1317914111

333

Peng, Z. ; Mcluckey, S. A. Int. J. Mass Spectrom. 2015 , 391 , 17 – 23 DOI: 10.1016/j.ijms.2015.07.027

334

Cotham, V. C. ; McGee, W. M. ; Brodbelt, J. S. Anal. Chem. 2016 , 88 , 8158 – 8165 DOI: 10.1021/acs.analchem.6b01901

335

Cotham, V. C. ; Shaw, J. B. ; Brodbelt, J. S. Anal. Chem. 2015 , 87 , 9396 – 9402 DOI: 10.1021/acs.analchem.5b02242

336

Coon, J. J. ; Shabanowitz, J. ; Hunt, D. F. ; Syka, J. E. P. J. Am. Soc. Mass Spectrom. 2005 , 16 , 880 – 882 DOI: 10.1016/j.jasms.2005.01.015

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336

Electron Transfer Dissociation of Peptide Anions

Coon, Joshua J.; Shabanowitz, Jeffrey; Hunt, Donald F.; Syka, John E. P.

Journal of the American Society for Mass Spectrometry (2005), 16 (6), 880-882CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)

Ion/ion reactions of multiply deprotonated peptide anions with xenon radical cations result in electron abstraction to generate charge-reduced peptide anions contg. a free-radical site. Peptide backbone cleavage then occurs by hydrogen radical abstraction from a backbone amide N to facilitate cleavage of the adjacent C-C bond, thereby producing a- and x-type product ions. Introduction of free-radical sites to multiply charged peptides allows access to new fragmentation pathways that are otherwise too costly (e.g., lowers activation energies). Further, ion/ion chem., namely electron transfer reactions, presents a rapid and efficient means of generating odd-electron multiply charged peptides; these reactions can be used for studying gas-phase chemistries and for peptide sequence anal.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXktlChtbc%253D&md5=8a78e22202feb6492b52e6e878ec0aea

337

Commodore, J. J. ; Cassady, C. J. J. Am. Soc. Mass Spectrom. 2016 , 27 , 1499 – 1509 DOI: 10.1007/s13361-016-1428-7

338

Commodore, J. J. ; Cassady, C. J. J. Mass Spectrom. 2017 , 52 , 218 – 229 DOI: 10.1002/jms.3919

339

Plummer, C. E. ; Stover, M. L. ; Bokatzian, S. S. ; Davis, J. T. M. ; Dixon, D. A. ; Cassady, C. J. J. Phys. Chem. B 2015 , 119 , 9661 – 9669 DOI: 10.1021/acs.jpcb.5b04486

340

McMillen, C. L. ; Wright, P. M. ; Cassady, C. J. J. Am. Soc. Mass Spectrom. 2016 , 27 , 847 – 855 DOI: 10.1007/s13361-016-1345-9

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340

Negative Ion In-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Sequencing Acidic Peptides

McMillen, Chelsea L.; Wright, Patience M.; Cassady, Carolyn J.

Journal of the American Society for Mass Spectrometry (2016), 27 (5), 847-855CODEN: JAMSEF; ISSN:1044-0305. (Springer)

Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the neg. ion mode on deprotonated peptides to det. its usefulness for obtaining extensive sequence information for acidic peptides. Eight biol. acidic peptides, ranging in size from 11 to 33 residues, were studied by neg. ion mode ISD (nISD). The matrixes 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was obsd. for every peptide except hirudin(54-65). Cleavage at the N-Cα bond of the peptide backbone, producing c' and z' ions, was dominant for all peptides. Cleavage of the N-Cα bond N-terminal to proline residues was not obsd. The formation of c and z ions is also found in electron transfer dissocn. (ETD), electron capture dissocn. (ECD), and pos. ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addn., spectra were obtained by pos. ion mode ISD for each protonated peptide; more sequence informative fragmentation was obsd. via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XisVGisbk%253D&md5=1700f5a3b325247065a2ffcb4e6b8fc2

341

McAlister, G. C. ; Russell, J. D. ; Rumachik, N. G. ; Hebert, A. S. ; Syka, J. E. P. ; Geer, L. Y. ; Westphall, M. S. ; Pagliarini, D. J. ; Coon, J. J. Anal. Chem. 2012 , 84 , 2875 – 2882 DOI: 10.1021/ac203430u

342

Rumachik, N. G. ; McAlister, G. C. ; Russell, J. D. ; Bailey, D. J. ; Wenger, C. D. ; Coon, J. J. J. Am. Soc. Mass Spectrom. 2012 , 23 , 718 – 727 DOI: 10.1007/s13361-011-0331-5

343

Shaw, J. B. ; Ledvina, A. R. ; Zhang, X. ; Julian, R. R. ; Brodbelt, J. S. J. Am. Chem. Soc. 2012 , 134 , 15624 – 15627 DOI: 10.1021/ja3032086

344

Shaw, J. B. ; Madsen, J. A. ; Xu, H. ; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 2012 , 23 , 1707 – 1715 DOI: 10.1007/s13361-012-0424-9

345

Shaw, J. B. ; Kaplan, D. A. ; Brodbelt, J. S. Anal. Chem. 2013 , 85 , 4721 – 4728 DOI: 10.1021/ac4005315

346

Riley, N. M. ; Rush, M. J. P. ; Rose, C. M. ; Richards, A. L. ; Kwiecien, N. W. ; Bailey, D. J. ; Hebert, A. S. ; Westphall, M. S. ; Coon, J. J. Mol. Cell. Proteomics 2015 , 14 , 2644 – 2660 DOI: 10.1074/mcp.M115.049726

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346

The Negative Mode Proteome with Activated Ion Negative Electron Transfer Dissociation (AI-NETD)

Riley, Nicholas M.; Rush, Matthew J. P.; Rose, Christopher M.; Richards, Alicia L.; Kwiecien, Nicholas W.; Bailey, Derek J.; Hebert, Alexander S.; Westphall, Michael S.; Coon, Joshua J.

Molecular & Cellular Proteomics (2015), 14 (10), 2644-2660CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

The field of proteomics almost uniformly relies on peptide cation anal., leading to an underrepresentation of acidic portions of proteomes, including relevant acidic posttranslational modifications. Despite the many benefits neg. mode proteomics can offer, peptide anion anal. remains in its infancy due mainly to challenges with high-pH reversed-phase sepns. and a lack of robust fragmentation methods suitable for peptide anion characterization. Here, we report the first implementation of activated ion neg. electron transfer dissocn. (AI-NETD) on the chromatog. timescale, generating 7,601 unique peptide identifications from Saccharomyces cerevisiae in single-shot nLC-MS/MS analyses of tryptic peptides-a greater than 5-fold increase over previous results with NETD alone. These improvements translate to identification of 1,106 proteins, making this work the first neg. mode study to identify more than 1,000 proteins in any system. We then compare the performance of AI-NETD for anal. of peptides generated by five proteases (trypsin, LysC, GluC, chymotrypsin, and AspN) for neg. mode analyses, identifying as many as 5,356 peptides (1,045 proteins) with LysC and 4,213 peptides (857 proteins) with GluC in yeast-characterizing 1,359 proteins in total. Finally, we present the first deep-sequencing approach for neg. mode proteomics, leveraging offline low-pH reversed-phase fractionation prior to online high-pH sepns. and peptide fragmentation with AI-NETD. With this platform, we identified 3,467 proteins in yeast with trypsin alone and characterized a total of 3,730 proteins using multiple proteases, or nearly 83% of the expressed yeast proteome. This work represents the most extensive neg. mode proteomics study to date, establishing AI-NETD as a robust tool for large-scale peptide anion characterization and making the neg. mode approach a more viable platform for future proteomic studies.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1GkurzK&md5=bca474d60ee8be84204bd4a7cbcb4f67

347

Riley, N. M. ; Bern, M. ; Westphall, M. S. ; Coon, J. J. J. Proteome Res. 2016 , 15 , 2768 – 2776 DOI: 10.1021/acs.jproteome.6b00319

[ACS Full Text ], [CAS], Google Scholar

347

Full-Featured Search Algorithm for Negative Electron-Transfer Dissociation

Riley, Nicholas M.; Bern, Marshall; Westphall, Michael S.; Coon, Joshua J.

Journal of Proteome Research (2016), 15 (8), 2768-2776CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Neg. electron-transfer dissocn. (NETD) has emerged as a premier tool for peptide anion anal., offering access to acidic post-translational modifications and regions of the proteome that are intractable with traditional pos.-mode approaches. Whole-proteome scale characterization is now possible with NETD, but proper informatic tools are needed to capitalize on advances in instrumentation. Currently only one database search algorithm (OMSSA) can process NETD data. Here we implement NETD search capabilities into the Byonic platform to improve the sensitivity of neg.-mode data analyses, and we benchmark these improvements using 90 min LC-MS/MS analyses of tryptic peptides from human embryonic stem cells. With this new algorithm for searching NETD data, we improved the no. of successfully identified spectra by as much as 80% and identified 8665 unique peptides, 24,639 peptide spectral matches, and 1338 proteins in activated-ion NETD analyses, more than doubling identifications from previous neg.-mode characterizations of the human proteome. Furthermore, we reanalyzed our recently published large-scale, multienzyme neg.-mode yeast proteome data, improving peptide and peptide spectral match identifications and considerably increasing protein sequence coverage. In all, we show that new informatics tools, in combination with recent advances in data acquisition, can significantly improve proteome characterization in neg.-mode approaches.

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348

Huzarska, M. ; Ugalde, I. ; Kaplan, D. A. ; Hartmer, R. ; Easterling, M. L. ; Polfer, N. C. Anal. Chem. 2010 , 82 , 2873 – 2878 DOI: 10.1021/ac9028592

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348

Negative Electron Transfer Dissociation of Deprotonated Phosphopeptide Anions: Choice of Radical Cation Reagent and Competition between Electron and Proton Transfer

Huzarska, Malwina; Ugalde, Israel; Kaplan, Desmond A.; Hartmer, Ralf; Easterling, Michael L.; Polfer, Nick C.

Analytical Chemistry (Washington, DC, United States) (2010), 82 (7), 2873-2878CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

Despite significant developments in mass spectrometry technol. in recent years, no routine proteomics sequencing tool is currently available for peptide anions. The use of a mol. open-shell cation is presented here as a possible reaction partner to induce electron transfer dissocn. with deprotonated peptide anions. In this neg. electron transfer dissocn. (NETD) scheme, an electron is abstracted from the peptide anion and transferred to the radical cation. This is demonstrated for the example of the fluoranthene cation, C16H10+·, which is reacted with deprotonated phosphorylated peptides in a 3-D ion trap mass spectrometer. Selective backbone cleavage at the Cα-C bond is obsd. to yield a and x fragments, similarly to electron detachment dissocn. (EDD) of peptide anions. Crucially, the phosphorylation site is left intact in the dissocn. process, allowing an identification and localization of the post-translational modification (PTM) site. In contrast, NETD using Xe+· as the reagent cation results in sequential neutral losses (CO2 and H3PO4) from a/x fragments, which complicate the interpretation of the mass spectra. This difference in dissocn. behavior can be understood in the framework of the reduced recombination energy of the electron transfer process for fluoranthene, which is estd. at 2.5-4.5 eV, compared to 6.7-8.7 eV for xenon. Similarly to ETD, proton transfer is found to compete with electron transfer processes in NETD. Isotope fitting of the charge-reduced species shows that in the case of fluoranthene-mediated NETD, proton transfer only accounts for <20%, whereas this process highly abundant for Xe+· (43 and 82%). Since proton abstraction from Xe+· is not possible, this suggests that Xe+· ionizes other transient species in the ion trap, which then engage in proton transfer reactions with the peptide anions.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXivV2mt7c%253D&md5=4a697aa4bfaae7438dd6af05915760b4

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Rush, M. J. P. ; Riley, N. M. ; Westphall, M. S. ; Syka, J. E. P. ; Coon, J. J. J. Am. Soc. Mass Spectrom. 2017 , 28 , 1324 – 1332 DOI: 10.1007/s13361-017-1600-8

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Yang, Y. S. ; Wang, C. C. ; Chen, B. H. ; Hou, Y. H. ; Hung, K. S. ; Mao, Y. C. Molecules 2015 , 20 , 2138 – 2164 DOI: 10.3390/molecules20022138

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350

Tyrosine sulfation as a protein post-translational modification

Yang, Yuh-Shyong; Wang, Chen-Chu; Chen, Bo-Han; Hou, You-Hua; Hung, Kuo-Sheng; Mao, Yi-Chih

Molecules (2015), 20 (2), 2138-2164/1-2138-2164/27, 27 pp.CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)

Integration of inorg. sulfate into biol. mols. plays an important role in biol. systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS) is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiol. conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST) through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biol. sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochem. information including the activity and the prepn. of TPST, methods for the detn. of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochem. and physiol. reactions.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXislWktLw%253D&md5=a2fe16243d8e46348c2713a30d1f1f5a

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Fuhs, S. R. ; Hunter, T. pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification Curr. Opin. Cell Biol. 2017 , 45 , 8 – 16 DOI: 10.1016/j.ceb.2016.12.010

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351

pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification

Fuhs, Stephen Rush; Hunter, Tony

Current Opinion in Cell Biology (2017), 45 (), 8-16CODEN: COCBE3; ISSN:0955-0674. (Elsevier Ltd.)

Histidine phosphorylation is crucial for prokaryotic signal transduction and as an intermediate for several metabolic enzymes, yet its role in mammalian cells remains largely uncharted. This is primarily caused by difficulties in studying histidine phosphorylation because of the relative instability of phosphohistidine (pHis) and lack of specific antibodies and methods to preserve and detect it. The recent synthesis of stable pHis analogs has enabled development of pHis-specific antibodies and their use has started to shed light onto this important, yet enigmatic posttranslational modification. We are beginning to understand that pHis has broader roles in protein and cellular function including; cell cycle regulation, phagocytosis, regulation of ion channel activity and metal ion coordination. Two mammalian histidine kinases (NME1 and NME2), two pHis phosphatases (PHPT1 and LHPP), and a handful of substrates were previously identified. These new tools have already led to the discovery of an addnl. phosphatase (PGAM5) and hundreds of putative substrates. New methodologies are also being developed to probe the pHis phosphoproteome and det. functional consequences, including neg. ion mode mass spectroscopy and unnatural amino acid incorporation. These new tools and strategies have the potential to overcome the unique challenges that have been holding back our understanding of pHis in cell biol.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVCgtLY%253D&md5=6b9b6f0c901bc0185395a0ad7bbd8d92

352

Gao, Y. ; McLuckey, S. A. Rapid Commun. Mass Spectrom. 2013 , 27 , 249 – 257 DOI: 10.1002/rcm.6428

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352

Electron transfer followed by collision-induced dissociation (NET-CID) for generating sequence information from backbone-modified oligonucleotide anions

Gao, Yang; McLuckey, Scott A.

Rapid Communications in Mass Spectrometry (2013), 27 (1), 249-257, S249/1-S249/9CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)

Oligonucleotides with 2'-modifications and/or phosphorothioate (PS) backbones are prone to undergo limited backbone fragmentation upon ion trap collision-induced dissocn. (CID). For better identification and characterization of chem. modified oligonucleotides, a more universal fragmentation method is desirable. Gas-phase dissocn. of various 2'-position-modified oligonucleotides and mixed-backbone oligonucleotides (MBOs) has been studied by ion trap CID of the radical anion species formed via electron transfer ion/ion reactions. For 2'-modified mix-mer radical anions, complete sequence information was generated with non-complementary d/w-ion series, while a/z-ions were obsd. randomly with relatively low intensity. The 2'-position modification, which has been obsd. to affect CID patterns of oligonucleotide anions, did not exhibit any observable influence on the dissocn. patterns of oligonucleotide radical anions. For MBOs comprised of DNA nucleotides, ion trap CID of even-electron species generated complementary a-B/w-type ions and multiple fragment types at the phosphorothioate (PS) linkages. For MBOs comprised of 2'-OMe-modified nucleotides, only PS bond cleavage was obsd. for ion trap CID of doubly deprotonated precursor ions. Neg. electron transfer reaction with or without supplemental activation of MBOs gave rise to a/d/w-type fragments similar to those of the 2'-modified mix-mers. PS bonds were obsd. to be more fragile under the electron detachment process, and phosphodiester (PO) bond cleavages were noted upon further collisional activation. NET-CID proved to be an efficient method of generating full sequence information for 2'-modifications and/or mixed-backbone oligonucleotides.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVCktrzL&md5=71d484b991d49ad8114070821cd4a025

353

Gao, Y. ; Yang, J. ; Cancilla, M. T. ; Meng, F. ; McLuckey, S. A. Anal. Chem. 2013 , 85 , 4713 – 4720 DOI: 10.1021/ac400448t

354

Gao, Y.; McLuckey, S. A. Fragmentation Reactions of Nucleic Acid Ions in the Gas Phase. In Nucleic Acids in the Gas Phase. Physical Chemistry in Action; Gabelica, V., Ed.; Springer: Berlin, Heidelberg, Germany, 2014 ; pp 131 – 182, DOI: DOI: 10.1007/978-3-642-54842-0_6 .

355

Wolff, J. J. ; Leach, F. E. ; Laremore, T. N. ; Kaplan, D. A. ; Easterling, M. L. ; Linhardt, R. J. ; Amster, I. J. ; Amster, I. J. Anal. Chem. 2010 , 82 , 3460 – 3466 DOI: 10.1021/ac100554a

356

Leach, F. E. ; Wolff, J. J. ; Xiao, Z. ; Ly, M. ; Laremore, T. N. ; Arungundram, S. ; Al-Mafraji, K. ; Venot, A. ; Boons, G.-J. ; Linhardt, R. J. ; Amster, I. J. ; Amster, I. J. Eur. Mass Spectrom. 2011 , 17 , 167 – 176 DOI: 10.1255/ejms.1120

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356

Negative electron transfer dissociation fourier transform mass spectrometry of glycosaminoglycan carbohydrates

Leach, Franklin E., III; Wolff, Jeremy J.; Xiao, Zhongping; Ly, Mellisa; Laremore, Tatiana N.; Arungundram, Sailaja; Al-Mafraji, Kanar; Venot, Andre; Boons, Geert-Jan; Linhardt, Robert J.; Amster, I. Jonathan

European Journal of Mass Spectrometry (2011), 17 (2), 167-176CODEN: EJMSCL; ISSN:1469-0667. (IM Publications)

Electron transfer through gas-phase ion-ion reactions has led to the widespread application of electron-based techniques once only capable in ion trapping mass spectrometers. Although any mass analyzer can, in theory, be coupled to an ion-ion reaction device (typically a 3-D ion trap), some systems of interest exceed the capabilities of most mass spectrometers. This case is particularly true in the structural characterization of glycosaminoglycan (GAG) oligosaccharides. To adequately characterize highly sulfated GAGs or oligosaccharides above the tetrasaccharide level, a high-resoln. mass analyzer is required. To extend previous efforts on an ion trap mass spectrometer, neg. electron transfer dissocn. coupled with a Fourier transform ion cyclotron resonance mass spectrometer has been applied to increasingly sulfated heparan sulfate and heparin tetrasaccharides as well as a dermatan sulfate octasaccharide. Results similar to those obtained by electron detachment dissocn. are obsd.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXpsVKlsLo%253D&md5=4962bfe3c707419edd895d0fa5795116

357

Huang, Y. ; Yu, X. ; Mao, Y. ; Costello, C. E. ; Zaia, J. ; Lin, C. Anal. Chem. 2013 , 85 , 11979 – 11986 DOI: 10.1021/ac402931j

358

Hu, H. ; Huang, Y. ; Mao, Y. ; Yu, X. ; Xu, Y. ; Liu, J. ; Zong, C. ; Boons, G.-J. ; Lin, C. ; Xia, Y. ; Zaia, J. Mol. Cell. Proteomics 2014 , 13 , 2490 – 2502 DOI: 10.1074/mcp.M114.039560

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358

A Computational Framework for Heparan Sulfate Sequencing Using High-resolution Tandem Mass Spectra

Hu, Han; Huang, Yu; Mao, Yang; Yu, Xiang; Xu, Yongmei; Liu, Jian; Zong, Chengli; Boons, Geert-Jan; Lin, Cheng; Xia, Yu; Zaia, Joseph

Molecular & Cellular Proteomics (2014), 13 (9), 2490-2502CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Heparan sulfate (HS) is a linear polysaccharide expressed on cell surfaces, in extracellular matrixes and cellular granules in metazoan cells. Through non-covalent binding to growth factors, morphogens, chemokines, and other protein families, HS is involved in all multicellular physiol. activities. Its biol. activities depend on the fine structures of its protein-binding domains, the detn. of which remains a daunting task. Methods have advanced to the point that mass spectra with information-rich product ions may be produced on purified HS saccharides. However, the interpretation of these complex product ion patterns has emerged as the bottleneck to the dissemination of these HS sequencing methods. To solve this problem, we designed HS-SEQ, the first comprehensive algorithm for HS de novo sequencing using high-resoln. tandem mass spectra. We tested HS-SEQ using neg. electron transfer dissocn. (NETD) tandem mass spectra generated from a set of pure synthetic saccharide stds. with diverse sulfation patterns. The results showed that HS-SEQ rapidly and accurately detd. the correct HS structures from large candidate pools.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVynurrL&md5=67deb76f56b8e5bb66ad1a234e28502f

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Leach, F. E. ; Riley, N. M. ; Westphall, M. S. ; Coon, J. J. ; Amster, I. J. J. Am. Soc. Mass Spectrom. 2017 , 28 , 1844 – 1854 DOI: 10.1007/s13361-017-1709-9

360

Merrill, A. E. ; Coon, J. J. Curr. Opin. Chem. Biol. 2013 , 17 , 779 – 786 DOI: 10.1016/j.cbpa.2013.06.011

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360

Quantifying proteomes and their post-translational modifications by stable isotope label-based mass spectrometry

Merrill, Anna E.; Coon, Joshua J.

Current Opinion in Chemical Biology (2013), 17 (5), 779-786CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)

A review. Stable isotope labeling coupled with mass spectrometry has revolutionized the scope and impact of protein expression studies. Label incorporation can occur metabolically or chem., and each method bears specific strengths and weaknesses. Quant. proteomics confidently identifies specific interactions between proteins and other biol. species, such as nucleic acids and metabolites. Extending label-based methods to phosphorylation-modified forms of proteins enables the construction of signaling networks and their temporal responses to stimuli. The integration of multiple data types offers systems-level insight on coordinated biol. processes. Finally, the development of methods applicable to tissue quantification suggests the emerging role of label-based, quant. mass spectrometry in translational science.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtVKnsLrJ&md5=5a399843250b10a1ccea234deeaa26ef

361

Hennrich, M. L. ; Marino, F. ; Groenewold, V. ; Kops, G. J. P. L. ; Mohammed, S. ; Heck, A. J. R. J. Proteome Res. 2013 , 12 , 2214 – 2224 DOI: 10.1021/pr400074f

[ACS Full Text ], [CAS], Google Scholar

361

Universal Quantitative Kinase Assay Based on Diagonal SCX Chromatography and Stable Isotope Dimethyl Labeling Provides High-definition Kinase Consensus Motifs for PKA and Human Mps1

Hennrich, Marco L.; Marino, Fabio; Groenewold, Vincent; Kops, Geert J. P. L.; Mohammed, Shabaz; Heck, Albert J. R.

Journal of Proteome Research (2013), 12 (5), 2214-2224CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

In order to understand cellular signaling, a clear understanding of kinase-substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatog. combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates in vitro and in vivo. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktF2msrs%253D&md5=4f39d0618d1b943ecdd4ff531c2933c1

362

Munoz, J. ; Low, T. Y. ; Kok, Y. J. ; Chin, A. ; Frese, C. K. ; Ding, V. ; Choo, A. ; Heck, A. J. R. Mol. Syst. Biol. 2011 , 7 , 550 DOI: 10.1038/msb.2011.84

363

Scholten, A. ; Mohammed, S. ; Low, T. Y. ; Zanivan, S. ; van Veen, T. A. B. ; Delanghe, B. ; Heck, A. J. R. Mol. Cell. Proteomics 2011 , 10 , O111.008474 DOI: 10.1074/mcp.O111.008474

364

Richards, A. L. ; Vincent, C. E. ; Guthals, A. ; Rose, C. M. ; Westphall, M. S. ; Bandeira, N. ; Coon, J. J. Mol. Cell. Proteomics 2013 , 12 , 3812 – 3823 DOI: 10.1074/mcp.M113.028951

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364

Neutron-encoded Signatures Enable Product Ion Annotation From Tandem Mass Spectra

Richards, Alicia L.; Vincent, Catherine E.; Guthals, Adrian; Rose, Christopher M.; Westphall, Michael S.; Bandeira, Nuno; Coon, Joshua J.

Molecular & Cellular Proteomics (2013), 12 (12), 3812-3823CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

We report the use of neutron-encoded (NeuCode) stable isotope labeling of amino acids in cell culture for the purpose of C-terminal product ion annotation. Two NeuCode labeling isotopologues of lysine, 13C615N2 and 2H8, which differ by 36 mDa, were metabolically embedded in a sample proteome, and the resultant labeled proteins were combined, digested, and analyzed via liq. chromatog. and mass spectrometry. With MS/MS scan resolving powers of ∼50,000 or higher, product ions contg. the C terminus (i.e. lysine) appear as a doublet spaced by exactly 36 mDa, whereas N-terminal fragments exist as a single m/z peak. Through theory and expt., we demonstrate that over 90% of all y-type product ions have detectable doublets. We report on an algorithm that can ext. these neutron signatures with high sensitivity and specificity. In other words, of 15,503 y-type product ion peaks, the y-type ion identification algorithm correctly identified 14,552 (93.2%) based on detection of the NeuCode doublet; 6.8% were misclassified (i.e. other ion types that were assigned as y-type products). Searching NeuCode labeled yeast with PepNovo+ resulted in a 34% increase in correct de novo identifications relative to searching through MS/MS only. We use this tool to simplify spectra prior to database searching, to sort unmatched tandem mass spectra for spectral richness, for correlation of co-fragmented ions to their parent precursor, and for de novo sequence identification.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFWrs7jJ&md5=c8ef822d42fe4d1964eadce12ca60d48

365

Rhoads, T. W. ; Rose, C. M. ; Bailey, D. J. ; Riley, N. M. ; Molden, R. C. ; Nestler, A. J. ; Merrill, A. E. ; Smith, L. M. ; Hebert, A. S. ; Westphall, M. S. ; Pagliarini, D. J. ; Garcia, B. A. ; Coon, J. J. Anal. Chem. 2014 , 86 , 2314 – 2319 DOI: 10.1021/ac403579s

366

Han, H. ; Pappin, D. J. ; Ross, P. L. ; McLuckey, S. A. J. Proteome Res. 2008 , 7 , 3643 – 3648 DOI: 10.1021/pr8001113

[ACS Full Text ], [CAS], Google Scholar

366

Electron Transfer Dissociation of iTRAQ Labeled Peptide Ions

Han, Hongling; Pappin, Darryl J.; Ross, Philip L.; McLuckey, Scott A.

Journal of Proteome Research (2008), 7 (9), 3643-3648CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Triply and doubly charged iTRAQ (isobaric tagging for relative and abs. quantitation) labeled peptide cations from a tryptic peptide mixt. of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications assocd. with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissocn. (ETD) of triply charged or activated ETD (ETD and supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addn. of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissocn. (CID) of the product ion at m/z 162 yielded the reporter ion at m/z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quant. information similar to that directly produced from CID. A 103 Da neutral loss species obsd. in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXovVWjtro%253D&md5=ba27d9178f68035d38ad6e42821c820e

367

Phanstiel, D. ; Unwin, R. ; McAlister, G. C. ; Coon, J. J. Anal. Chem. 2009 , 81 , 1693 – 1698 DOI: 10.1021/ac8019202

368

McAlister, G. C. ; Huttlin, E. L. ; Haas, W. ; Ting, L. ; Jedrychowski, M. P. ; Rogers, J. C. ; Kuhn, K. ; Pike, I. ; Grothe, R. A. ; Blethrow, J. D. ; Gygi, S. P. Anal. Chem. 2012 , 84 , 7469 – 7478 DOI: 10.1021/ac301572t

369

Hebert, A. S. ; Merrill, A. E. ; Bailey, D. J. ; Still, A. J. ; Westphall, M. S. ; Strieter, E. R. ; Pagliarini, D. J. ; Coon, J. J. Nat. Methods 2013 , 10 , 332 – 334 DOI: 10.1038/nmeth.2378

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369

Neutron-encoded mass signatures for multiplexed proteome quantification

Hebert, Alexander S.; Merrill, Anna E.; Bailey, Derek J.; Still, Amelia J.; Westphall, Michael S.; Strieter, Eric R.; Pagliarini, David J.; Coon, Joshua J.

Nature Methods (2013), 10 (4), 332-334CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

We describe a protein quantification method called neutron encoding that exploits the subtle mass differences caused by nuclear binding energy variation in stable isotopes. These mass differences are synthetically encoded into amino acids and incorporated into yeast and mouse proteins via metabolic labeling. Mass spectrometry anal. with high mass resoln. (>200,000) reveals the isotopologue-embedded peptide signals, permitting quantification. Neutron encoding will enable highly multiplexed proteome anal. with excellent dynamic range and accuracy.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXivFOrtbw%253D&md5=fe452869f46445e15fea1d9ba68ce193

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Yu, Q. ; Shi, X. ; Feng, Y. ; Kent, K. C. ; Li, L. Anal. Chim. Acta 2017 , 968 , 40 – 49 DOI: 10.1016/j.aca.2017.03.003

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370

Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

Yu, Qing; Shi, Xudong; Feng, Yu; Kent, K. Craig; Li, Lingjun

Analytica Chimica Acta (2017), 968 (), 40-49CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)

Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissocn. (CID) and higher-energy collisional dissocn. (HCD), isobaric tagging technique could also work with electron-transfer dissocn. (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addn., common fragmentation mechanism in ETD results in altered reporter ion prodn., decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed di-Me leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissocn. (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quant. proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion prodn. Biol. studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quant. proteomics and quant. PTM studies.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXls1Gqt7Y%253D&md5=b52580c450bd09ff56a010c19e30da3e

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Chalkley, R. J. ; Medzihradszky, K. F. ; Lynn, A. J. ; Baker, P. R. ; Burlingame, A. L. Anal. Chem. 2010 , 82 , 579 – 584 DOI: 10.1021/ac9018582

372

Li, W. ; Song, C. ; Bailey, D. J. ; Tseng, G. C. ; Coon, J. J. ; Wysocki, V. H. Anal. Chem. 2011 , 83 , 9540 – 9545 DOI: 10.1021/ac202327r

373

Askenazi, M. ; Bandeira, N. ; Chalkley, R. J. ; Deutsch, E. ; Lam, H. H. N. ; McDonald, W. H. ; Neubert, T. ; Rudnick, P. A. ; Martens, L. J. Biomol. Technol. 2011 , 22 , S20

374

Tyanova, S. ; Temu, T. ; Cox, J. Nat. Protoc. 2016 , 11 , 2301 – 2319 DOI: 10.1038/nprot.2016.136

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374

The MaxQuant computational platform for mass spectrometry-based shotgun proteomics

Tyanova, Stefka; Temu, Tikira; Cox, Juergen

Nature Protocols (2016), 11 (12), 2301-2319CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)

MaxQuant is one of the most frequently used platforms for mass-spectrometry (MS)-based proteomics data anal. Since its first release in 2008, it has grown substantially in functionality and can be used in conjunction with more MS platforms. Here we present an updated protocol covering the most important basic computational workflows, including those designed for quant. label-free proteomics, MS1-level labeling and isobaric labeling techniques. This protocol presents a complete description of the parameters used in MaxQuant, as well as of the configuration options of its integrated search engine, Andromeda. This protocol update describes an adaptation of an existing protocol that substantially modifies the technique. Important concepts of shotgun proteomics and their implementation in MaxQuant are briefly reviewed, including different quantification strategies and the control of false-discovery rates (FDRs), as well as the anal. of post-translational modifications (PTMs). The MaxQuant output tables, which contain information about quantification of proteins and PTMs, are explained in detail. Furthermore, we provide a short version of the workflow that is applicable to data sets with simple and std. exptl. designs. The MaxQuant algorithms are efficiently parallelized on multiple processors and scale well from desktop computers to servers with many cores. The software is written in C# and is freely available at http://www.maxquant.org.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslynsL7O&md5=31539b285b373b7fcb4e6a497857d228

375

Röst, H. L. ; Sachsenberg, T. ; Aiche, S. ; Bielow, C. ; Weisser, H. ; Aicheler, F. ; Andreotti, S. ; Ehrlich, H.-C. ; Gutenbrunner, P. ; Kenar, E. ; Liang, X. ; Nahnsen, S. ; Nilse, L. ; Pfeuffer, J. ; Rosenberger, G. ; Rurik, M. ; Schmitt, U. ; Veit, J. ; Walzer, M. ; Wojnar, D. ; Wolski, W. E. ; Schilling, O. ; Choudhary, J. S. ; Malmström, L. ; Aebersold, R. ; Reinert, K. ; Kohlbacher, O. Nat. Methods 2016 , 13 , 741 – 748 DOI: 10.1038/nmeth.3959

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375

OpenMS: a flexible open-source software platform for mass spectrometry data analysis

Rost, Hannes L.; Sachsenberg, Timo; Aiche, Stephan; Bielow, Chris; Weisser, Hendrik; Aicheler, Fabian; Andreotti, Sandro; Ehrlich, Hans-Christian; Gutenbrunner, Petra; Kenar, Erhan; Liang, Xiao; Nahnsen, Sven; Nilse, Lars; Pfeuffer, Julianus; Rosenberger, George; Rurik, Marc; Schmitt, Uwe; Veit, Johannes; Walzer, Mathias; Wojnar, David; Wolski, Witold E.; Schilling, Oliver; Choudhary, Jyoti S.; Malmstrom, Lars; Aebersold, Ruedi; Reinert, Knut; Kohlbacher, Oliver

Nature Methods (2016), 13 (9), 741-748CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

High-resoln. mass spectrometry (MS) has become an important tool in the life sciences, contributing to the diagnosis and understanding of human diseases, elucidating biomol. structural information and characterizing cellular signaling networks. However, the rapid growth in the vol. and complexity of MS data makes transparent, accurate and reproducible anal. difficult. We present OpenMS 2.0 (http://www.openms.de), a robust, open-source, cross-platform software specifically designed for the flexible and reproducible anal. of high-throughput MS data. The extensible OpenMS software implements common mass spectrometric data processing tasks through a well-defined application programming interface in C++ and Python and through standardized open data formats. OpenMS addnl. provides a set of 185 tools and ready-made workflows for common mass spectrometric data processing tasks, which enable users to perform complex quant. mass spectrometric analyses with ease.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1ejtLrF&md5=6185e304e7a051764414f932c0c266aa

376

Hunt, D. F. ; Shabanowitz, J. ; Bai, D. L. J. Am. Soc. Mass Spectrom. 2015 , 26 , 1256 – 1258 DOI: 10.1007/s13361-015-1105-2

377

Zolg, D. P. ; Wilhelm, M. ; Schnatbaum, K. ; Zerweck, J. ; Knaute, T. ; Delanghe, B. ; Bailey, D. J. ; Gessulat, S. ; Ehrlich, H.-C. ; Weininger, M. ; Yu, P. ; Schlegl, J. ; Kramer, K. ; Schmidt, T. ; Kusebauch, U. ; Deutsch, E. W. ; Aebersold, R. ; Moritz, R. L. ; Wenschuh, H. ; Moehring, T. ; Aiche, S. ; Huhmer, A. ; Reimer, U. ; Kuster, B. Nat. Methods 2017 , 14 , 259 – 262 DOI: 10.1038/nmeth.4153

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377

Building ProteomeTools based on a complete synthetic human proteome

Zolg, Daniel P.; Wilhelm, Mathias; Schnatbaum, Karsten; Zerweck, Johannes; Knaute, Tobias; Delanghe, Bernard; Bailey, Derek J.; Gessulat, Siegfried; Ehrlich, Hans-Christian; Weininger, Maximilian; Yu, Peng; Schlegl, Judith; Kramer, Karl; Schmidt, Tobias; Kusebauch, Ulrike; Deutsch, Eric W.; Aebersold, Ruedi; Moritz, Robert L.; Wenschuh, Holger; Moehring, Thomas; Aiche, Stephan; Huhmer, Andreas; Reimer, Ulf; Kuster, Bernhard

Nature Methods (2017), 14 (3), 259-262CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

We describe ProteomeTools, a project building mol. and digital tools from the human proteome to facilitate biomedical research. Here we report the generation and multimodal liq. chromatog.-tandem mass spectrometry anal. of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at http://www.proteometools.org) will be extended to >1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvV2hs74%253D&md5=38886bd3f60f51777db50bf17d3cb162

378

Good, D. M. ; Wenger, C. D. ; McAlister, G. C. ; Bai, D. L. ; Hunt, D. F. ; Coon, J. J. J. Am. Soc. Mass Spectrom. 2009 , 20 , 1435 – 1440 DOI: 10.1016/j.jasms.2009.03.006

379

Good, D. M. ; Wenger, C. D. ; Coon, J. J. Proteomics 2010 , 10 , 164 – 167 DOI: 10.1002/pmic.200900570

380

Sridhara, V. ; Bai, D. L. ; Chi, A. ; Shabanowitz, J. ; Hunt, D. F. ; Bryant, S. H. ; Geer, L. Y. Proteome Sci. 2012 , 10 , 8 DOI: 10.1186/1477-5956-10-8

[Crossref], [PubMed], [CAS], Google Scholar

380

Increasing peptide identifications and decreasing search times for ETD spectra by pre-processing and calculation of parent precursor charge

Sridhara, Viswanadham; Bai, Dina L.; Chi, An; Shabanowitz, Jeffrey; Hunt, Donald F.; Bryant, Stephen H.; Geer, Lewis Y.

Proteome Science (2012), 10 (), 8CODEN: PSRCCC; ISSN:1477-5956. (BioMed Central Ltd.)

Background: Electron Transfer Dissocn. [ETD] can dissoc. multiply charged precursor polypeptides, providing extensive peptide backbone cleavage. ETD spectra contain charge reduced precursor peaks, usually of high intensity, and whose pattern is dependent on its parent precursor charge. These charge reduced precursor peaks and assocd. neutral loss peaks should be removed before these spectra are searched for peptide identifications. ETD spectra can also contain ion-types other than c and z. Modifying search strategies to accommodate these ion-types may aid in increased peptide identifications. Addnl., if the precursor mass is measured using a lower resoln. instrument such as a linear ion trap, the charge of the precursor is often not known, reducing sensitivity and increasing search times. We implemented algorithms to remove these precursor peaks, accommodate new ion-types in noise filtering routine in OMSSA and to est. any unknown precursor charge, using Linear Discriminant Anal. [LDA]. Results: Spectral pre-processing to remove precursor peaks and their assocd. neutral losses prior to protein sequence library searches resulted in a 9.8% increase in peptide identifications at a 1% False Discovery Rate [FDR] compared to previous OMSSA filter. Modifications to the OMSSA noise filter to accommodate various ion-types resulted in a further 4.2% increase in peptide identifications at 1% FDR. Moreover, ETD spectra when searched with charge states obtained from the precursor charge detn. algorithm is shown to be up to 3.5 times faster than the general range search method, with a minor 3.8% increase in sensitivity. Conclusion: Overall, there is an 18.8% increase in peptide identifications at 1% FDR by incorporating the new precursor filter, noise filter and by using the charge detn. algorithm, when compared to previous versions of OMSSA.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmtVOks74%253D&md5=49b36cce99dcbaef10afc172815e34e4

381

Xia, Q. ; Lee, M. V. ; Rose, C. M. ; Marsh, A. J. ; Hubler, S. L. ; Wenger, C. D. ; Coon, J. J. J. Am. Soc. Mass Spectrom. 2011 , 22 , 255 – 264 DOI: 10.1007/s13361-010-0029-0

382

Chi, H. ; Chen, H. ; He, K. ; Wu, L. ; Yang, B. ; Sun, R.-X. ; Liu, J. ; Zeng, W.-F. ; Song, C.-Q. ; He, S.-M. ; Dong, M.-Q. J. Proteome Res. 2013 , 12 , 615 – 625 DOI: 10.1021/pr3006843

[ACS Full Text ], [CAS], Google Scholar

382

pNovo+: De Novo Peptide Sequencing Using Complementary HCD and ETD Tandem Mass Spectra

Chi, Hao; Chen, Haifeng; He, Kun; Wu, Long; Yang, Bing; Sun, Rui-Xiang; Liu, Jianyun; Zeng, Wen-Feng; Song, Chun-Qing; He, Si-Min; Dong, Meng-Qiu

Journal of Proteome Research (2013), 12 (2), 615-625CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

De novo peptide sequencing is the only tool for extg. peptide sequences directly from tandem mass spectrometry (MS) data without any protein database. However, neither the accuracy nor the efficiency of de novo sequencing has been satisfactory, mainly due to incomplete fragmentation information in exptl. spectra. Recent advancement in MS technol. has enabled acquisition of higher energy collisional dissocn. (HCD) and electron transfer dissocn. (ETD) spectra of the same precursor. These spectra contain complementary fragmentation information and can be collected with high resoln. and high mass accuracy. Taking these advantages, we have developed a new algorithm called pNovo+, which greatly improves the accuracy and speed of de novo sequencing. On tryptic peptides, 86% of the topmost candidate sequences deduced by pNovo+ from HCD + ETD spectral pairs matched the database search results, and the success rate reached 95% if the top three candidates were included, which was much higher than using only HCD (87%) or only ETD spectra (57%). On Asp-N, Glu-C, or Elastase digested peptides, 69-87% of the HCD + ETD spectral pairs were correctly identified by pNovo+ among the topmost candidates, or 84-95% among the top three. On av., it takes pNovo+ only 0.018 s to ext. the sequence from a spectrum or spectral pair on a common personal computer. This is more than three times as fast as other de novo sequencing programs. The increase of speed is mainly due to pDAG, a component algorithm of pNovo+. pDAG finds the k longest paths in a directed acyclic graph without the antisymmetry restriction. We have verified that the antisymmetry restriction is unnecessary for high resoln., high mass accuracy data. The extensive use of HCD and ETD spectral information and the pDAG algorithm make pNovo+ an excellent de novo sequencing tool.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjslen&md5=8227a30e5ede5b796ec33a9b711498f6

383

Yan, Y. ; Kusalik, A. J. ; Wu, F.-X. IEEE Trans. Nanobioscience 2015 , 14 , 478 – 484 DOI: 10.1109/TNB.2015.2419194

384

Yan, Y. ; Zhang, K. BMC Bioinf. 2016 , 17 , 538 DOI: 10.1186/s12859-016-1330-0

[Crossref], [PubMed], [CAS], Google Scholar

384

Spectra library assisted de novo peptide sequencing for HCD and ETD spectra pairs

Yan Yan; Zhang Kaizhong

BMC bioinformatics (2016), 17 (Suppl 17), 538 ISSN:.

BACKGROUND: De novo peptide sequencing via tandem mass spectrometry (MS/MS) has been developed rapidly in recent years. With the use of spectra pairs from the same peptide under different fragmentation modes, performance of de novo sequencing is greatly improved. Currently, with large amount of spectra sequenced everyday, spectra libraries containing tens of thousands of annotated experimental MS/MS spectra become available. These libraries provide information of the spectra properties, thus have the potential to be used with de novo sequencing to improve its performance. RESULTS: In this study, an improved de novo sequencing method assisted with spectra library is proposed. It uses spectra libraries as training datasets and introduces significant scores of the features used in our previous de novo sequencing method for HCD and ETD spectra pairs. Two pairs of HCD and ETD spectral datasets were used to test the performance of the proposed method and our previous method. The results show that this proposed method achieves better sequencing accuracy with higher ranked correct sequences and less computational time. CONCLUSIONS: This paper proposed an advanced de novo sequencing method for HCD and ETD spectra pair and used information from spectra libraries and significant improved previous similar methods.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1c3isFGhtA%253D%253D&md5=8458b8d3bd6c37fd88d1dd04df4a8820

385

Yan, Y. ; Kusalik, A. J. ; Wu, F.-X. IEEE/ACM Trans. Comput. Biol. Bioinf. 2017 , 14 , 337 – 344 DOI: 10.1109/TCBB.2015.2389813

[Crossref], [PubMed], [CAS], Google Scholar

385

NovoExD: De novo Peptide Sequencing for ETD/ECD Spectra

Yan Yan; Kusalik Anthony J; Wu Fang-Xiang

IEEE/ACM transactions on computational biology and bioinformatics (2017), 14 (2), 337-344 ISSN:.

De novo peptide sequencing using tandem mass spectrometry (MS/MS) data has become a major computational method for sequence identification in recent years. With the development of new instruments and technology, novel computational methods have emerged with enhanced performance. However, there are only a few methods focusing on ECD/ETD spectra, which mainly contain variants of c -ions and z-ions. Here, a de novo sequencing method for ECD/ETD spectra, NovoExD, is presented. NovoExD applies a new form of spectrum graph with multiple edge types (called a GMET), considers multiple peptide tags, and integrates amino acid combination (AAC) and fragment ion charge information. Its performance is compared with another successful de novo sequencing method, pNovo+, which has an option for ECD/ETD spectra. Experiments conducted on three different datasets show that the average full length peptide identification accuracy of NovoExD is as high as 88.70 percent, and that NovoExD's average accuracy is more than 20 percent greater on all datasets than that of pNovo+.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cvjslKksQ%253D%253D&md5=0dbb666adb57edd879770e6f7b89b5c3

386

Zhu, Z. ; Su, X. ; Go, E. P. ; Desaire, H. Anal. Chem. 2014 , 86 , 9212 – 9219 DOI: 10.1021/ac502176n

387

Mayampurath, A. ; Yu, C.-Y. ; Song, E. ; Balan, J. ; Mechref, Y. ; Tang, H. Anal. Chem. 2014 , 86 , 453 – 463 DOI: 10.1021/ac402338u

388

Lee, L. Y. ; Moh, E. S. X. ; Parker, B. L. ; Bern, M. ; Packer, N. H. ; Thaysen-Andersen, M. J. Proteome Res. 2016 , 15 , 3904 – 3915 DOI: 10.1021/acs.jproteome.6b00438

[ACS Full Text ], [CAS], Google Scholar

388

Toward Automated N-Glycopeptide Identification in Glycoproteomics

Lee, Ling Y.; Moh, Edward S. X.; Parker, Benjamin L.; Bern, Marshall; Packer, Nicolle H.; Thaysen-Andersen, Morten

Journal of Proteome Research (2016), 15 (10), 3904-3915CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

Advances in software-driven glycopeptide identification have facilitated N-glycoproteomics studies reporting thousands of intact N-glycopeptides, i.e., N-glycan-conjugated peptides, but the automated identification process remains to be scrutinized. Herein, the authors compare the site-specific glycoprofiling efficiency of the PTM-centric search engine Byonic relative to manual expert annotation using typical glycoproteomics acquisition and data anal. strategies, but of a single glycoprotein, the uncharacterized multiple N-glycosylated human basigin. Detailed site-specific ref. glycoprofiles of purified basigin were manually established using ion trap CID-MS/MS and high-resoln. Q-Exactive Orbitrap HCD-MS/MS of tryptic N-glycopeptides and released N-glycans. The micro- and macro-heterogeneous basigin N-glycosylation was site-specifically glycoprofiled using Byonic with or without a background of complex peptides using Q-Exactive Orbitrap HCD-MS/MS. The automated glycoprofiling efficiencies were assessed against the site-specific ref. glycoprofiles and target/decoy proteome databases. Within the limits of this single glycoprotein anal., the search criteria and confidence thresholds (Byonic scores) recommended by the vendor provided high glycoprofiling accuracy and coverage (both >80%) and low peptide FDRs (<1%). The data complexity, search parameters including search space (proteome/glycome size), mass tolerance and peptide modifications, and confidence thresholds affected the automated glycoprofiling efficiency and anal. time. Correct identification of ambiguous peptide modifications (methionine oxidn./carbamidomethylation) whose mass differences coincide with several monosaccharide mass differences (Fuc/Hex/HexNAc) and of ambiguous isobaric (Hex1NeuAc1-R/Fuc1NeuGc1-R) or near-isobaric (NeuAc1-R/Fuc2-R) monosaccharide sub-compns. remain challenging in automated glycoprofiling, arguing particular attention to N-glycopeptides displaying such "difficult-to-identify" features. This study provides valuable insights into the automated glycopeptide identification process, stimulating further developments in FDR-based glycoproteomics.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtlaktb7P&md5=05c49ed66635928fd4d55cbe9d470206

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Darula, Z. ; Chalkley, R. J. ; Lynn, A. ; Baker, P. R. ; Medzihradszky, K. F. Amino Acids 2011 , 41 , 321 – 328 DOI: 10.1007/s00726-010-0692-2

390

Zhu, Z. ; Su, X. ; Clark, D. F. ; Go, E. P. ; Desaire, H. Anal. Chem. 2013 , 85 , 8403 – 8411 DOI: 10.1021/ac401814h

391

Chalkley, R. J. ; Baker, P. R. Anal. Bioanal. Chem. 2017 , 409 , 571 – 577 DOI: 10.1007/s00216-016-9981-2

[Crossref], [PubMed], [CAS], Google Scholar

391

Use of a glycosylation site database to improve glycopeptide identification from complex mixtures

Chalkley, Robert J.; Baker, Peter R.

Analytical and Bioanalytical Chemistry (2017), 409 (2), 571-577CODEN: ABCNBP; ISSN:1618-2642. (Springer)

New mass spectrometry instrumentation, particularly those with electron transfer dissocn. fragmentation, has made the anal. of complex glycopeptide mixts. accessible. However, software tools need to be optimized for interpretation of this type of data. Glycopeptide identification is challenging due to the no. of different peptide and sugar moieties that can be combined, leading to a large no. of potential compns. to consider. In this manuscript, different strategies for reducing the no. of peptides and glycopeptides considered in database searching are compared. Adaptation of the software Protein Prospector to support the use of a ref. modification site database doubled the no. of glycopeptide IDs. The potential of this as an improved anal. strategy is discussed.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1eltbnE&md5=ef3fe8a2ccabe8844dc9a0e3e84b442c

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Fellers, R. T. ; Greer, J. B. ; Early, B. P. ; Yu, X. ; LeDuc, R. D. ; Kelleher, N. L. ; Thomas, P. M. Proteomics 2015 , 15 , 1235 – 1238 DOI: 10.1002/pmic.201400313

[Crossref], [PubMed], [CAS], Google Scholar

392

ProSight Lite: Graphical software to analyze top-down mass spectrometry data

Fellers, Ryan T.; Greer, Joseph B.; Early, Bryan P.; Yu, Xiang; LeDuc, Richard D.; Kelleher, Neil L.; Thomas, Paul M.

Proteomics (2015), 15 (7), 1235-1238CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)

Many top-down proteomics expts. focus on identifying and localizing PTMs and other potential sources of "mass shift" on a known protein sequence. A simple application to match ion masses and facilitate the iterative hypothesis testing of PTM presence and location would assist with the data anal. in these expts. ProSight Lite is a free software tool for matching a single candidate sequence against a set of mass spectrometric observations. Fixed or variable modifications, including both PTMs and a select no. of glycosylations, can be applied to the amino acid sequence. The application reports multiple scores and a matching fragment list. Fragmentation maps can be exported for publication in either portable network graphic (PNG) or scalable vector graphic (SVG) format. ProSight Lite can be freely downloaded from , installs and updates from the web, and requires Windows 7 or a higher version.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvVamtrw%253D&md5=757b65a0b15b103c511c9abf3bea87b4

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Cai, W. ; Guner, H. ; Gregorich, Z. R. ; Chen, A. J. ; Ayaz-Guner, S. ; Peng, Y. ; Valeja, S. G. ; Liu, X. ; Ge, Y. Mol. Cell. Proteomics 2016 , 15 , 703 – 714 DOI: 10.1074/mcp.O115.054387

[Crossref], [PubMed], [CAS], Google Scholar

393

MASH Suite Pro: A Comprehensive Software Tool for Top-Down Proteomics

Cai, Wenxuan; Guner, Huseyin; Gregorich, Zachery R.; Chen, Albert J.; Ayaz-Guner, Serife; Peng, Ying; Valeja, Santosh G.; Liu, Xiaowen; Ge, Ying

Molecular & Cellular Proteomics (2016), 15 (2), 703-714CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)

Top-down mass spectrometry (MS)-based proteomics is arguably a disruptive technol. for the comprehensive anal. of all proteoforms arising from genetic variation, alternative splicing, and posttranslational modifications (PTMs). However, the complexity of top-down high-resoln. mass spectra presents a significant challenge for data anal. Herein, we have developed MASH Suite Pro, a comprehensive software tool for top-down proteomics with multifaceted functionality. MASH Suite Pro is capable of processing high-resoln. MS and tandem MS (MS/MS) data using two deconvolution algorithms to optimize protein identification results. In addn., MASH Suite Pro allows for the characterization of PTMs and sequence variations, as well as the relative quantitation of multiple proteoforms in different exptl. conditions. The program also provides visualization components for validation and correction of the computational outputs. Furthermore, MASH Suite Pro facilitates data reporting and presentation via direct output of the graphics. Thus, MASH Suite Pro significantly simplifies and speeds up the interpretation of high-resoln. top-down proteomics data by integrating tools for protein identification, quantitation, characterization, and visual validation into a customizable and user-friendly interface. We envision that MASH Suite Pro will play an integral role in advancing the burgeoning field of top-down proteomics.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslOkt70%253D&md5=09915b4ab4fb6b413771912a2be68daa

394

Zhou, M. ; Paša-Tolić, L. ; Stenoien, D. L. J. Proteome Res. 2017 , 16 , 599 – 608 DOI: 10.1021/acs.jproteome.6b00694

[ACS Full Text ], [CAS], Google Scholar

394

Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry

Zhou, Mowei; Pasa-Tolic, Ljiljana; Stenoien, David L.

Journal of Proteome Research (2017), 16 (2), 599-608CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

As histones play central roles in most chromosomal functions including regulation of DNA replication, DNA damage repair, and gene transcription, both their basic biol. and their roles in disease development have been the subject of intense study. Since multiple PTMs along the entire protein sequence are potential regulators of histones, a top-down approach, where intact proteins are analyzed, is ultimately required for complete characterization of proteoforms. However, significant challenges remain for top-down histone anal. primarily because of deficiencies in sepn./resolving power and effective identification algorithms. Here, the authors used state of the art mass spectrometry and a bioinformatics workflow for targeted data anal. and visualization. The workflow uses ProMex for intact mass deconvolution, MSPathFinder as search engine, and LcMsSpectator as a data visualization tool. When complemented with the open-modification tool TopPIC, this workflow enabled identification of novel histone PTMs including tyrosine bromination on histone H4 and H2A, H3 glutathionylation, and mapping of conventional PTMs along the entire protein for many histone subunits.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvF2msrrL&md5=ee5793b3c4dd6df22836fd1eecf41bcd

395

Xiao, K. ; Yu, F. ; Tian, Z. J. Proteomics 2017 , 152 , 41 – 47 DOI: 10.1016/j.jprot.2016.10.010

[Crossref], [PubMed], [CAS], Google Scholar

395

Top-down protein identification using isotopic envelope fingerprinting

Xiao, Kaijie; Yu, Fan; Tian, Zhixin

Journal of Proteomics (2017), 152 (), 41-47CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)

For top-down protein database search and identification from tandem mass spectra, our isotopic envelope fingerprinting search algorithm and ProteinGoggle search engine have demonstrated their strength of efficiently resolving heavily overlapping data as well sepg. non-ideal data with non-ideal isotopic envelopes from ideal ones with ideal isotopic envelopes. Here we report our updated ProteinGoggle 2.0 for intact protein database search with full-capacity. The indispensable updates include users' optional definition of dynamic post-translational modifications and static chem. labeling during database creation, comprehensive dissocn. methods and ion series, as well as a Proteoform Score for each proteoform. ProteinGoggle has previously been benchmarked with both collision-based dissocn. (CID, HCD) and electron-based dissocn. (ETD) data of either intact proteins or intact proteomes. Here we report our further benchmarking of the new version of ProteinGoggle with publically available photon-based dissocn. (UVPD) data (http://hdl.handle.net/2022/17316) of intact E. coli ribosomal proteins. Protein species (aka proteoforms) function at their mol. level, and diverse structures and biol. roles of every proteoform come from often co-occurring proteolysis, amino acid variation and post-translational modifications. Complete and high-throughput capture of this combinatorial information of proteoforms has become possible in evolving top-down proteomics; yet, various methods and technologies, esp. database search and bioinformatics identification tools, in the top-down pipeline are still in their infancy stages and demand intensive research and development.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVagsr3K&md5=9abd9ff1cb2c1018a4d0470644fd0b40

396

Li, L. ; Tian, Z. Rapid Commun. Mass Spectrom. 2013 , 27 , 1267 – 1277 DOI: 10.1002/rcm.6565

[Crossref], [PubMed], [CAS], Google Scholar

396

Interpreting raw biological mass spectra using isotopic mass-to-charge ratio and envelope fingerprinting

Li, Li; Tian, Zhixin

Rapid Communications in Mass Spectrometry (2013), 27 (11), 1267-1277CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)

RATIONALE : Soft ionization, high-resoln. mass spectrometry is widely used to characterize large biol. mols., such as proteins. Deconvolution ('deisotoping') of isotopic envelopes (iEs) in biol. mass spectra into monoisotopic or av. masses is challenging due to low signals and heavily overlapped iEs, resulting in many wrong interpretations. METHODS : Isotopic envelopes (iEs) are directly used without deisotoping to identify biol. mols. An algorithm, isotopic mass-to-charge ratio (m/z) and envelope fingerprinting (iMEF), was implemented in the ProteinGoggle search engine for top-down intact protein database searching. iMEF combines isotopic m/z fingerprinting (iMF) and isotopic envelop fingerprinting (iEF), where 'Isotopic mass-to-charge ratio' means the m/z value of the most abundant isotopic peak within the iE of a precursor or product ion. iMF is used to 'fish' precursor or product ion candidates from the database, which is pre-built and contains all iE information (precursor and product ions) of all proteoforms of the studied system. iEF identifies matching precursor or product ions. A protein is finally identified with user-specified total no. of matching product ions and post-translational modification scores. RESULTS : The working principles of iMEF and ProteinGoggle, and the definition of a set of related parameters and scoring metrics, are illustrated with high-resoln. tandem mass spectrometric anal. of a mixt. of ubiquitin and the HUMAN histone H4 proteoforms. Ubiquitin was confidently identified from its CID, ETD, and HCD spectra with 57, 91, and 66 matching product ions, resp.; 125 proteoforms were confidently found from the H4 dataset. The locations of PTMs in 54 and 6 isoforms were partially and fully identified. CONCLUSIONS : Database search with iMEF bypasses 'deisotoping' avoiding assocd. errors, and also provides full quality control of matching precursor and product ions and finally protein IDs. Overlapped iEs of different product ions could also be confidently unwrapped in situ. Improvement and addn. of more functionalities and utilities of ProteinGoggle are underway.

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397

LeDuc, R. D. ; Fellers, R. T. ; Early, B. P. ; Greer, J. B. ; Thomas, P. M. ; Kelleher, N. L. J. Proteome Res. 2014 , 13 , 3231 – 3240 DOI: 10.1021/pr401277r

[ACS Full Text ], [CAS], Google Scholar

397

The C-Score: A Bayesian Framework to Sharply Improve Proteoform Scoring in High-Throughput Top Down Proteomics

LeDuc, Richard D.; Fellers, Ryan T.; Early, Bryan P.; Greer, Joseph B.; Thomas, Paul M.; Kelleher, Neil L.

Journal of Proteome Research (2014), 13 (7), 3231-3240CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

The automated processing of data generated by top down proteomics would benefit from improved scoring for protein identification and characterization of highly related protein forms (proteoforms). Here the authors propose the "C-score" (short for Characterization Score), a Bayesian approach to the proteoform identification and characterization problem, implemented within a framework to allow the infusion of expert knowledge into generative models that take advantage of known properties of proteins and top down anal. systems (e.g., fragmentation propensities, "off-by-1 Da" discontinuous errors, and intelligent weighting for site-specific modifications). The performance of the scoring system based on the initial generative models was compared to the current probability-based scoring system used within both ProSightPC and ProSightPTM on a manually curated set of 295 human proteoforms. The current implementation of the C-score framework generated a marked improvement over the existing scoring system as measured by the area under the curve on the resulting ROC chart (AUC of 0.99 vs. 0.78).

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398

Lermyte, F. ; Łacki, M. K. ; Valkenborg, D. ; Baggerman, G. ; Gambin, A. ; Sobott, F. Int. J. Mass Spectrom. 2015 , 390 , 146 – 154 DOI: 10.1016/j.ijms.2015.08.008

399

Ciach, M. A. ; Łącki, M. K. ; Miasojedow, B. ; Lermyte, F. ; Valkenborg, D. ; Sobott, F. ; Gambin, A. J. Comput. Biol. 2017 , 96 – 107 DOI: 10.1089/cmb.2017.0156

400

Liu, X. ; Dekker, L. J. M. ; Wu, S. ; Vanduijn, M. M. ; Luider, T. M. ; Tolić, N. ; Kou, Q. ; Dvorkin, M. ; Alexandrova, S. ; Vyatkina, K. ; Paša-Tolić, L. ; Pevzner, P. A. J. Proteome Res. 2014 , 13 , 3241 – 3248 DOI: 10.1021/pr401300m

[ACS Full Text ], [CAS], Google Scholar

400

De Novo Protein Sequencing by Combining Top-Down and Bottom-Up Tandem Mass Spectra

Liu, Xiaowen; Dekker, Lennard J. M.; Wu, Si; Van Duijn, Martijn M.; Luider, Theo M.; Tolic, Nikola; Kou, Qiang; Dvorkin, Mikhail; Alexandrova, Sonya; Vyatkina, Kira; Pasa-Tolic, Ljiljana; Pevzner, Pavel A.

Journal of Proteome Research (2014), 13 (7), 3241-3248CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

There are two approaches for de novo protein sequencing: Edman degrdn. and mass spectrometry (MS). Existing MS-based methods characterize a novel protein by assembling tandem mass spectra of overlapping peptides generated from multiple proteolytic digestions of the protein. Because each tandem mass spectrum covers only a short peptide of the target protein, the key to high coverage protein sequencing is to find spectral pairs from overlapping peptides in order to assemble tandem mass spectra to long ones. However, overlapping regions of peptides may be too short to be confidently identified. High-resoln. mass spectrometers have become accessible to many labs. These mass spectrometers are capable of analyzing mols. of large mass values, boosting the development of top-down MS. Top-down tandem mass spectra cover whole proteins. However, top-down tandem mass spectra, even combined, rarely provide full ion fragmentation coverage of a protein. We propose an algorithm, TBNovo, for de novo protein sequencing by combining top-down and bottom-up MS. In TBNovo, a top-down tandem mass spectrum is utilized as a scaffold, and bottom-up tandem mass spectra are aligned to the scaffold to increase sequence coverage. Expts. on data sets of two proteins showed that TBNovo achieved high sequence coverage and high sequence accuracy.

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401

Vyatkina, K. ; Wu, S. ; Dekker, L. J. M. ; VanDuijn, M. M. ; Liu, X. ; Tolić, N. ; Dvorkin, M. ; Alexandrova, S. ; Luider, T. M. ; Paša-Tolić, L. ; Pevzner, P. A. J. Proteome Res. 2015 , 14 , 4450 – 4462 DOI: 10.1021/pr501244v

[ACS Full Text ], [CAS], Google Scholar

401

De Novo Sequencing of Peptides from Top-Down Tandem Mass Spectra

Vyatkina, Kira; Wu, Si; Dekker, Lennard J. M.; VanDuijn, Martijn M.; Liu, Xiaowen; Tolic, Nikola; Dvorkin, Mikhail; Alexandrova, Sonya; Luider, Theo M.; Pasa-Tolic, Ljiljana; Pevzner, Pavel A.

Journal of Proteome Research (2015), 14 (11), 4450-4462CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)

De novo sequencing of proteins and peptides is one of the most important problems in mass spectrometry-driven proteomics. A variety of methods have been developed to accomplish this task from a set of bottom-up tandem (MS/MS) mass spectra. However, a more recently emerged top-down technol., now gaining more and more popularity, opens new perspectives for protein anal. and characterization, implying a need for efficient algorithms to process this kind of MS/MS data. Here, we describe a method that allows for the retrieval, from a set of top-down MS/MS spectra, of long and accurate sequence fragments of the proteins contained in the sample. To this end, we outline a strategy for generating high-quality sequence tags from top-down spectra, and introduce the concept of a T-Bruijn graph by adapting to the case of tags the notion of an A-Bruijn graph widely used in genomics. The output of the proposed approach represents the set of amino acid strings spelled out by optimal paths in the connected components of a T-Bruijn graph. We illustrate its performance on top-down data sets acquired from carbonic anhydrase 2 (CAH2) and the Fab region of alemtuzumab.

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Where Does Electron Transfer Dissociation Occur in an Orbitrap

Source: https://pubs.acs.org/doi/10.1021/acs.analchem.7b04810

Where Does Electron Transfer Dissociation Occur in an Orbitrap

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