Abstract
Ubiquitination has emerged as one of the major post-translational modifications that decide on protein fate, targeting, and regulation of protein function. Whereas the ubiquitination of proteins can be monitored with classic biochemical methods, the mapping of modified side chains proves to be challenging. More recently, mass spectrometry has been applied to identify ubiquitinated proteins and also their sites of modification. Typically, liquid chromatography tandem mass spectrometry (LC-MS/MS) based approaches, including collision-induced fragmentation (CID), have been successfully used in the past. However, a potential difficulty arises from the unstable nature of this modification, and also that the isopeptide bond linkage between C-terminal glycine and the N(ε) lysyl side chain is susceptible to fragmentation under these conditions. Here we investigate the utility of electron-transfer dissociation (ETD)-based fragmentation to detect ubiquitination sites in proteins. Our results indicate that ETD can provide alternative fragmentation patterns that allow detection of gly-gly-modified lysyl side chains, in particular z+1 fragment ions derived from triply charged precursor ions. We subsequently applied ETD fragmentation-based analysis and detected novel ubiquitination sites on DNA polymerase B1 that were not easily observed using CID. We conclude that ETD can provide significant alternative fragmentation information that complements CID-derived data to improve the coverage when mapping ubiquitination sites in proteins.
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Kerscher, O.; Felberbaum, R.; Hochstrasser, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell. Dev. Biol. 2006, 22, 159–180.
Ravid, T.; Hochstrasser, M. Diversity of degradation signals in the ubiquitin-proteasome system. Nat. Rev. Mol. Cell. Biol. 2008, 9(9), 679–690.
Sowa, M. E.; Harper, J. W. From loops to chains: Unraveling the mysteries of polyubiquitin chain specificity and processivity. ACS Chem. Biol. 2006, 1(1), 20–24.
Newton, K.; Matsumoto, M. L.; Wertz, I. E.; Kirkpatrick, D. S.; Lill, J. R.; Tan, J.; Dugger, D.; Gordon, N.; Sidhu, S. S.; Fellouse, F. A.; Komuves, L.; French, D. M.; Ferrando, R. E.; Lam, C.; Compaan, D.; Yu, C.; Bosanac, I.; Hymowitz, S. G.; Kelley, R. F.; Dixit, V. M. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 2008, 134(4), 668–678.
Denison, C.; Kirkpatrick, D. S.; Gygi, S. P. Proteomic insights into ubiquitin and ubiquitin-like proteins. Curr. Opin. Chem. Biol. 2005, 9(1), 69–75.
Kirkpatrick, D. S.; Denison, C.; Gygi, S. P. Weighing in on ubiquitin: The expanding role of mass-spectrometry-based proteomics. Nat. Cell. Biol. 2005, 7(8), 750–757.
Peng, J.; Schwartz, D.; Elias, J. E.; Thoreen, C. C.; Cheng, D.; Marsischky, G.; Roelofs, J.; Finley, D.; Gygi, S. P. A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 2003, 21(8), 921–926.
Peng, J. Evaluation of proteomic strategies for analyzing ubiquitinated proteins. BMB Rep. 2008, 41(3), 177–183.
Bennett, E. J.; Shaler, T. A.; Woodman, B.; Ryu, K. Y.; Zaitseva, T. S.; Becker, C. H.; Bates, G. P.; Schulman, H.; Kopito, R. R. Global changes to the ubiquitin system in Huntington’s disease. Nature 2007, 448(7154), 704–708.
Kirkpatrick, D. S.; Hathaway, N. A.; Hanna, J.; Elsasser, S.; Rush, J.; Finley, D.; King, R. W.; Gygi, S. P. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nat. Cell. Biol. 2006, 8(7), 700–710.
Mikesh, L. M.; Ueberheide, B.; Chi, A.; Coon, J. J.; Syka, J. E.; Shabanowitz, J.; Hunt, D. F. The utility of ETD mass spectrometry in proteomic analysis. Biochim. Biophys. Acta. 2006, 1764(12), 1811–1822.
Syka, J. E.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2004, 101(26), 9528–9533.
Udeshi, N. D.; Compton, P. D.; Shabanowitz, J.; Hunt, D. F.; Rose, K. L. Methods for analyzing peptides and proteins on a chromatographic timescale by electron-transfer dissociation mass spectrometry. Nat. Protoc. 2008, 3(11), 1709–1717.
Udeshi, N. D.; Shabanowitz, J.; Hunt, D. F.; Rose, K. L. Analysis of proteins and peptides on a chromatographic timescale by electron-transfer dissociation MS. FEBS J. 2007, 274(24), 6269–6276.
Wu, S. L.; Huhmer, A. F.; Hao, Z.; Karger, B. L. 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. J. Proteome Res. 2007, 6(11), 4230–4244.
Good, D. M.; Wirtala, M.; McAlister, G. C.; Coon, J. J. Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteom. 2007, 6(11), 1942–1951.
Steen, H.; Mann, M. The ABC’s (and XYZ’s) of peptide sequencing. Nat. Rev. Mol. Cell. Biol. 2004, 5(9), 699–711.
Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. Electron capture dissociation of multiply charged protein cations: A nonergodic process. J. Am. Chem. Soc. 1998, 120, 3265–3266.
Li, X.; Cournoyer, J. J.; Lin, C.; O’Connor, P. B. The effect of fixed charge modifications on electron capture dissociation. J Am. Soc. Mass Spectrom. 2008, 19(10), 1514–1526.
Hartmer, R.; Kaplan, D. A.; Gebhardt, C. R.; Ledertheil, T.; Brekenfeld, A. Multiple ion/ion reactions in the 3D ion trap: Selective reagent anion production for ETD and PTR from a single compound. Int. J. Mass Spectrom. 2008, 276, 82–90.
Kinter, M.; Sherman, N. E. Protein sequencing and identification using tandem mass spectrometry; John Wiley & Sons: New York, 2000; pp. 147–164.
Parsons, J. L.; Tait, P. S.; Finch, D.; Dianova, I. I.; Allinson, S. L.; Dianov, G. L. CHIP-mediated degradation and DNA damage-dependent stabilization regulate base excision repair proteins. Mol. Cell. 2008, 29(4), 477–487.
Batycka, M.; Inglis, N. F.; Cook, K.; Adam, A.; Fraser-Pitt, D.; Smith, D. G.; Main, L.; Lubben, A.; Kessler, B. M. Ultra-fast tandem mass spectrometry scanning combined with monolithic column liquid chromatography increases throughput in proteomic analysis. Rapid Commun. Mass Spectrom. 2006, 20(14), 2074–2080.
Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20(18), 3551–3567.
Taylor, G. K.; Goodlett, D. R. Rules governing protein identification by mass spectrometry. Rapid Commun. Mass Spectrom. 2005, 19(23), 3420.
Piotrowski, J.; Beal, R.; Hoffman, L.; Wilkinson, K. D.; Cohen, R. E.; Pickart, C. M. Inhibition of the 26 S proteasome by polyubiquitin chains synthesized to have defined lengths. J Biol. Chem. 1997, 272(38), 23712–23721.
Biemann, K. Appendix 5. Nomenclature for peptide fragment ions (positive ions). Methods Enzymol 1990, 193, 886–887.
Wiesner, J.; Premsler, T.; Sickmann, A. Application of electron transfer dissociation (ETD) for the analysis of posttranslational modifications. Proteomics 2008, 8(21), 4466–4483.
Cooper, H. J.; Case, M. A.; McLendon, G. L.; Marshall, A. G. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of metal-ion selected dynamic protein libraries. J. Am. Chem. Soc. 2003, 125(18), 5331–5339.
Baba, T.; Satake, H.; Manri, N.; Hasegawa, H. Electron capture dissociation in a radio-frequency linear ion trap. NanoFrontier 2009, NF/TN(E)002, 1–4.
Pitteri, S. J.; Chrisman, P. A.; Hogan, J. M.; McLuckey, S. A. Electron transfer ion/ion reactions in a three-dimensional quadrupole ion trap: Reactions of doubly and triply protonated peptides with SO2*. Anal. Chem. 2005, 77(6), 1831–1839.
Kaplan, D. A.; Hartmer, R.; Speir, J. P.; Stoermer, C.; Gumerov, D.; Easterling, M. L.; Brekenfeld, A.; Kim, T.; Laukien, F.; Park, M. A. Electron transfer dissociation in the hexapole collision cell of a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer. Rapid Commun. Mass Spectrom. 2008, 22(3), 271–278.
McAlister, G. C.; Berggren, W. T.; Griep-Raming, J.; Horning, S.; Makarov, A.; Phanstiel, D.; Stafford, G.; Swaney, D. L.; Syka, J. E.; Zabrouskov, V.; Coon, J. J. A proteomics grade electron transfer dissociation-enabled hybrid linear ion trap-Orbitrap mass spectrometer. J. Proteome Res. 2008, 7(8), 3127–3136.
Stoermer, C.; Hartmer, R.; Lubeck, M.; Schneider, A.; Kaplan, D. A.; Park, M. A. Automated ETD of large peptides and medium size proteins in a QTOF on LC time scale. ABRF Symposium; Memphis, February, 2009.
Brown, J. M.; Campuzano, I.; Pringle, S.; Chapman, R. Electron transfer dissociation with a rf traveling ion guide collision cell of a QTOF. Proceedings of the 56th ASMS Conference on Mass Spectrometry and Allied Topics; Denver, CO, June, 2008.
Cooper, H. J.; Health, J. K.; Jaffray, E.; Hay, R. T.; Lam, T. T.; Marshall, A. G. Identification of sites of ubiquitination in proteins: A Fourier transform ion cyclotron resonance mass spectrometry approach. Anal. Chem. 2004, 76(23), 6982–6988.
Nielsen, M. L.; Vermeulen, M.; Bonaldi, T.; Cox, J.; Moroder, L.; Mann, M. Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat. Methods 2008, 5(6), 459–460.
Coon, J. J. A supplemental activation method for high efficiency electron transfer dissociation of doubly protonated peptide precursors. Anal. Chem. 2007, 79(2), 477–485.
Colinge, J.; Masselot, A.; Giron, M.; Dessingy, T.; Magnin, J. OLAV: Towards high-throughput tandem mass spectrometry data identification. Proteomics 2003, 3(8), 1454–1463.
Geer, L. Y.; Markey, S. P.; Kowalak, J. A.; Wagner, L.; Xu, M.; Maynard, D. M.; Yang, X.; Shi, W.; Bryant, S. H. Open mass spectrometry search algorithm. J. Proteome Res. 2004, 3(5), 958–964.
Schaefer, H.; Chamrad, D. C.; Herrmann, M.; Stuwe, J.; Becker, G.; Klose, J.; Blueggel, M.; Meyer, H. E.; Marcus, K. Study of posttranslational modifications in lenticular αA-Crystallin of mice using proteomic analysis techniques. Biochim. Biophys. Acta 2006, 1764(12), 1948–1962.
Parker, C. E.; Warren, M. R.; Mocanu, V.; Greer, S. F.; Borchers, C. H. Mass spectrometric determination of protein ubiquitination. Methods Mol. Biol. 2008, 446, 109–130.
Parker, C. E.; Mocanu, V.; Warren, M. R.; Greer, S. F.; Borchers, C. H. Mass spectrometric determination of protein ubiquitination. Methods Mol. Biol. 2005, 301, 153–173.
Taouatas, N.; Drugan, M. M.; Heck, A. J.; Mohammed, S. Straightforward ladder sequencing of peptides using a Lys-N metalloendopeptidase. Nat. Methods 2008, 5(5), 405–407.
Mollah, S.; Wertz, I. E.; Phung, Q.; Arnott, D.; Dixit, V. M.; Lill, J. R. Targeted mass spectrometric strategy for global mapping of ubiquitination on proteins. Rapid Commun. Mass Spectrom. 2007, 21(20), 3357–3364.
Xu, P.; Peng, J. Characterization of polyubiquitin chain structure by middle-down mass spectrometry. Anal. Chem. 2008, 80(9), 3438–4344.
Meinnel, T.; Serero, A.; Giglione, C. Impact of the N-terminal amino acid on targeted protein degradation. Biol. Chem. 2006, 387(7), 839–851.
Ravid, T.; Hochstrasser, M. Autoregulation of an E2 enzyme by ubiquitin-chain assembly on its catalytic residue. Nat. Cell. Biol. 2007, 9(4), 422–427.
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Published online May 18, 2009
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Sobott, F., Watt, S.J., Smith, J. et al. Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination. J Am Soc Mass Spectrom 20, 1652–1659 (2009). https://doi.org/10.1016/j.jasms.2009.04.023
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DOI: https://doi.org/10.1016/j.jasms.2009.04.023