Abstract
Tandem mass spectra (MS/MS) produced using electron transfer dissociation (ETD) differ from those derived from collision-activated dissociation (CAD) in several important ways. Foremost, the predominant fragment ion series are different: c- and z ·-type ions are favored in ETD spectra while b- and y-type ions comprise the bulk of the fragments in CAD spectra. Additionally, ETD spectra possess charge-reduced precursors and unique neutral losses. Most database search algorithms were designed to analyze CAD spectra, and have only recently been adapted to accommodate c- and z ·-type ions; therefore, inclusion of these additional spectral features can hinder identification, leading to lower confidence scores and decreased sensitivity. Because of this, it is important to pre-process spectral data before submission to a database search to remove those features that cause complications. Here, we demonstrate the effects of removing these features on the number of unique peptide identifications at a 1% false discovery rate (FDR) using the open mass spectrometry search algorithm (OMSSA). When analyzing two biologic replicates of a yeast protein extract in three total analyses, the number of unique identifications with a ∼1% FDR increased from 4611 to 5931 upon spectral pre-processing—an increase of ∼28. 6%. We outline the most effective pre-processing methods, and provide free software containing these algorithms.
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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(13), 3265–3266.
Coon, J. J.; Ueberheide, B.; Syka, J. E.; Dryhurst, D. D.; Ausio, J.; Shabanowitz, J.; Hunt, D. F. Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 2005, 102(27), 9463–9468.
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.
Molina, H.; Horn, D. M.; Tang, N.; Mathivanan, S.; Pandey, A. Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 2007, 104(7), 2199–2204.
Chi, A.; Huttenhower, C.; Geer, L. Y.; Coon, J. J.; Syka, J. E. P.; Bai, D. L.; Shabanowitz, J.; Burke, D. J.; Troyanskaya, O. G.; Hunt, D. F. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 2007, 104(7), 2193–2198.
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.
Huang, Y. Y.; Triscari, J. M.; Tseng, G. C.; Pasa-Tolic, L.; Lipton, M. S.; Smith, R. D.; Wysocki, V. H. Statistical characterization of the charge state and residue dependence of low-energy CID peptide dissociation patterns. Anal. Chem. 2005, 77(18), 5800–5813.
Cooper, H. J.; Hakansson, K.; Marshall, A. G.; Hudgins, R. R.; Haselmann, K. F.; Kjeldsen, F.; Budnik, B. A.; Polfer, N. C.; Zubarev, R. A. Letter: The diagnostic value of amino acid side-chain losses in electron capture dissociation of polypeptides: Comment on: “Can the (M(.)-X) region in electron capture dissociation provide reliable information on amino acid composition of polypeptides?” Eur. J. Mass Spectrom. 2002, 8, 461–469 (2002). Eur. J. Mass Spectrom. (Chichester) 2003, 9(3), 221–222.
Cooper, H. J.; Hudgins, R. R.; Hakansson, K.; Marshall, A. G. Characterization of amino acid side chain losses in electron capture dissociation. J. Am. Soc. Mass Spectrom. 2002, 13(3), 241–249.
Fung, Y. M. E.; Chan, T. W. D. Experimental and theoretical investigations of the loss of amino acid side chains in electron capture dissociation of model peptides. J. Am. Soc. Mass Spectrom. 2005, 16(9), 1523–1535.
Falth, M.; Savitski, M. M.; Nielsen, M. L.; Kjeldsen, F.; Andren, P. E.; Zubarev, R. A. Analytical utility of small neutral losses from reduced species in electron capture dissociation studied using SwedECD database. Anal. Chem. 2008, 80(21), 8089–8094.
Savitski, M. M.; Nielsen, M. L.; Zubarev, R. A. Side-chain losses in electron capture dissociation to improve peptide identification. Anal. Chem. 2007, 79(6), 2296–2302.
Geer, L. Y.; Markey, S. P.; Kowalak, J. A.; Wagner, L.; Xu, M.; Maynard, D. M.; Yang, X. Y.; Shi, W. Y.; Bryant, S. H. Open mass spectrometry search algorithm. J. Proteome Res. 2004, 3(5), 958–964.
Swaney, D. L.; McAlister, G. C.; Coon, J. J. Decision tree-driven tandem mass spectrometry for shotgun proteomics. Nat. Methods. 2008, 5(11), 959–964.
Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P. Large-scale phosphorylation analysis of mouse liver. Proc. Natl. Acad. Sci. U. S. A. 2007, 104(5), 1488–1493.
Martin, S. E.; Shabanowitz, J.; Hunt, D. F.; Marto, J. A. Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 2000, 72(18), 4266–4274.
McAlister, G. C.; Berggren, W. T.; Griep-Raming, J.; Horning, S.; Makarov, A.; Phanstiel, D.; Stafford, G.; Swaney, D. L.; Syka, J. E. P.; 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.
McAlister, G. C.; Phanstiel, D.; Good, D. M.; Berggren, W. T.; Coon, J. J. Implementation of electron-transfer dissociation on a hybrid linear ion trap-orbitrap mass spectrometer. Anal. Chem. 2007, 79(10), 3525–3534.
Elias, J. E.; Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods. 2007, 4(3), 207–214.
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Published online March 14, 2009
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Good, D.M., Wenger, C.D., McAlister, G.C. et al. Post-acquisition ETD spectral processing for increased peptide identifications. J Am Soc Mass Spectrom 20, 1435–1440 (2009). https://doi.org/10.1016/j.jasms.2009.03.006
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DOI: https://doi.org/10.1016/j.jasms.2009.03.006