Post-acquisition ETD spectral processing for increased peptide identifications

  • David M. Good
  • Craig D. Wenger
  • Graeme C. McAlister
  • Dina L. Bai
  • Donald F. Hunt
  • Joshua J. Coon
Focus: The Orbitrap

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.

References

  1. 1.
    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.CrossRefGoogle Scholar
  2. 2.
    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.CrossRefGoogle Scholar
  3. 3.
    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.CrossRefGoogle Scholar
  4. 4.
    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.CrossRefGoogle Scholar
  5. 5.
    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.CrossRefGoogle Scholar
  6. 6.
    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.CrossRefGoogle Scholar
  7. 7.
    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.CrossRefGoogle Scholar
  8. 8.
    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.CrossRefGoogle Scholar
  9. 9.
    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.CrossRefGoogle Scholar
  10. 10.
    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.CrossRefGoogle Scholar
  11. 11.
    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.CrossRefGoogle Scholar
  12. 12.
    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.CrossRefGoogle Scholar
  13. 13.
    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.CrossRefGoogle Scholar
  14. 14.
    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.CrossRefGoogle Scholar
  15. 15.
    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.CrossRefGoogle Scholar
  16. 16.
    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.CrossRefGoogle Scholar
  17. 17.
    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.CrossRefGoogle Scholar
  18. 18.
    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.CrossRefGoogle Scholar
  19. 19.
    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.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2009

Authors and Affiliations

  • David M. Good
    • 1
  • Craig D. Wenger
    • 1
  • Graeme C. McAlister
    • 1
  • Dina L. Bai
    • 2
  • Donald F. Hunt
    • 2
    • 3
  • Joshua J. Coon
    • 1
    • 4
  1. 1.Department of ChemistryUniversity of WisconsinMadisonUSA
  2. 2.Department of ChemistryUniversity of VirginiaCharlottesvilleUSA
  3. 3.Department of PathologyUniversity of VirginiaCharlottesvilleUSA
  4. 4.Department of Biomolecular ChemistryUniversity of WisconsinMadisonUSA

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