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Decision tree–driven tandem mass spectrometry for shotgun proteomics

Abstract

Mass spectrometry has become a key technology for modern large-scale protein sequencing. Tandem mass spectrometry, the process of peptide ion dissociation followed by mass-to-charge ratio (m/z) analysis, is the critical component for peptide identification. Recent advances in mass spectrometry now permit two discrete, and complementary, types of peptide ion fragmentation: collision-activated dissociation (CAD) and electron transfer dissociation (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 addition, the DT method also identified 7,422 phosphopeptides, compared to either 2,801 (CAD) or 5,874 (ETD) phosphopeptides.

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Figure 1: Comprehensive probability of a high-confidence peptide identification.
Figure 2: Overlap between various combinations of duplicate analyses of yeast peptides.
Figure 3: Schematic representation of the probabilistic decision tree.
Figure 4: Distribution of identified peptides using CAD, ETD or DT methods.
Figure 5: Overlap between various combinations of duplicate analyses of hESC phosphopeptides.

References

  1. Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).

    CAS  Article  Google Scholar 

  2. Hunt, D.F., Yates, J.R., Shabanowitz, J., Winston, S. & Hauer, C.R. Protein sequencing by tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 83, 6233–6237 (1986).

    CAS  Article  Google Scholar 

  3. Coon, J.J., Syka, J.E.P., Shabanowitz, J. & Hunt, D.F. Tandem mass spectrometry for peptide and protein sequence analysis. Biotechniques 38, 519–523 (2005).

    CAS  Article  Google Scholar 

  4. Washburn, M.P., Wolters, D. & Yates, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247 (2001).

    CAS  Article  Google Scholar 

  5. Peng, J., Elias, J.E., Thoreen, C.C., Licklider, L.J. & Gygi, S.P. Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J. Proteome Res. 2, 43–50 (2003).

    CAS  Article  Google Scholar 

  6. de Godoy, L.M.F. et al. Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system. Genome Biol. 7, R50 (2006).

    Article  Google Scholar 

  7. Good, D.M., Wirtala, M., McAlister, G.C. & Coon, J.J. Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteomics 6, 1942–1951 (2007).

    CAS  Article  Google Scholar 

  8. Dongre, A.R., Jones, J.L., Somogyi, A. & Wysocki, V.H. Influence of peptide composition, gas-phase basicity, and chemical modification on fragmentation efficiency: Evidence for the mobile proton model. J. Am. Chem. Soc. 118, 8365–8374 (1996).

    CAS  Article  Google Scholar 

  9. Huang, Y. et al. Statistical characterization of the charge state and residue dependence of low-energy CID peptide dissociation patterns. Anal. Chem. 77, 5800–5813 (2005).

    CAS  Article  Google Scholar 

  10. Coon, J.J., Syka, J.E.P., Schwartz, J.C., Shabanowitz, J. & Hunt, D.F. Anion dependence in the partitioning between proton and electron transfer in ion/ion reactions. Int. J. Mass Spectrom. 236, 33–42 (2004).

    CAS  Article  Google Scholar 

  11. Syka, J.E.P., 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. USA 101, 9528–9533 (2004).

    CAS  Article  Google Scholar 

  12. Coon, J.J. et al. Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 102, 9463–9468 (2005).

    CAS  Article  Google Scholar 

  13. Hogan, J.M., Pitteri, S.J., Chrisman, P.A. & McLuckey, S.A. Complementary structural information from a tryptic N-linked glycopeptide via electron transfer ion/ion reactions and collision-induced dissociation. J. Proteome Res. 4, 628–632 (2005).

    CAS  Article  Google Scholar 

  14. 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. 77, 1831–1839 (2005).

    CAS  Article  Google Scholar 

  15. Good, D.M. & Coon, J.J. Advancing proteomics with ion/ion chemistry. Biotechniques 40, 783–789 (2006).

    CAS  Article  Google Scholar 

  16. Chi, A. et al. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc. Natl. Acad. Sci. USA 104, 2193–2198 (2007).

    CAS  Article  Google Scholar 

  17. Khidekel, N. et al. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat. Chem. Biol. 3, 339–348 (2007).

    CAS  Article  Google Scholar 

  18. Lecchi, S. et al. Tandem phosphorylation of Ser-911 and Thr-912 at the C terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activation. J. Biol. Chem. 282, 35471–35481 (2007).

    CAS  Article  Google Scholar 

  19. Schwartz, J.C., Senko, M.W. & Syka, J.E.P. A two-dimensional quadrupole ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 13, 659–669 (2002).

    CAS  Article  Google Scholar 

  20. Xia, Y. et al. Implementation of ion/ion reactions in a quadrupole/time-of-flight tandem mass spectrometer. Anal. Chem. 78, 4146–4154 (2006).

    CAS  Article  Google Scholar 

  21. 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. 79, 3525–3534 (2007).

    CAS  Article  Google Scholar 

  22. McAlister, G.C. et al. A proteomics grade electron transfer dissociation-enabled hybrid linear ion trap-orbitrap mass spectrometer. J. Proteome Res. 7, 3127–3136 (2008).

    CAS  Article  Google Scholar 

  23. Kaplan, D.A. et al. Electron transfer dissociation in the hexapole collision cell of a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer. Rapid. Commun. Mass. Spectrom. 22, 271–278 (2008).

    CAS  Article  Google Scholar 

  24. Schroeder, M.J., Shabanowitz, J., Schwartz, J.C., Hunt, D.F. & Coon, J.J. A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. Anal. Chem. 76, 3590–3598 (2004).

    CAS  Article  Google Scholar 

  25. Beausoleil, S.A. et al. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. USA 101, 12130–12135 (2004).

    CAS  Article  Google Scholar 

  26. Villen, J., Beausoleil, S.A., Gerber, S.A. & Gygi, S.P. Large-scale phosphorylation analysis of mouse liver. Proc. Natl. Acad. Sci. USA 104, 1488–1493 (2007).

    CAS  Article  Google Scholar 

  27. Good, D.M., Wirtala, M., McAlister, G.C. & Coon, J.J. Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell Proteomics 6, 1942–1951 (2007).

    CAS  Article  Google Scholar 

  28. Molina, H., Matthiesen, R., Kandasamy, K. & Pandey, A. Comprehensive comparison of collision induced dissociation and electron transfer dissociation. Anal. Chem. 80, 4825–4835 (2008).

    CAS  Article  Google Scholar 

  29. Swaney, D.L. et al. Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors. Anal. Chem. 79, 477–485 (2007).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank J. Griep-Raming, S. Horning, O. Lange, A. Makarov, J. Schwartz, M. Senko, G. Stafford and J. Syka (all at Thermo Scientific) for providing advice and technical assistance during the development of the ETD-Orbitrap instrumentation. We also thank B. Craig, S. Herbst, H. Coon and K. Thompson for growing and collecting the yeast, and J. Antosiewicz-Bourget and J. Thomson for growing the hESCs. We acknowledge J. Marto and S. Ficarro for advice with the Waters chromatography system. We thank G. Barrett-Wilt, D. Good, J. Keith, D. Phanstiel and A. Huhmer for helpful discussions. The University of Wisconsin, the Beckman Foundation, Eli Lilly and the US National Institutes of Health (NIH) (1R01GM080148 to J.J.C.) provided financial support for this work. G.C.M. and D.L.S. acknowledge support from the NIH predoctoral fellowships (Biotechnology Training Program, NIH 5T32GM08349 to G.C.M.; the Genomic Sciences Training Program, NIH 5T32HG002706 to D.L.S.).

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Authors and Affiliations

Authors

Contributions

G.C.M., D.L.S. and J.J.C. designed research; G.C.M. and D.L.S. performed research; G.C.M. modified the instrument; and G.C.M., D.L.S. and J.J.C. wrote the paper.

Note: Supplementary information is available on the Nature Methods website.

Corresponding author

Correspondence to Joshua J Coon.

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Competing interests

J.J.C. is a co-inventor on two US patent applications related, in part, to the material presented here.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 1, Supplementary Methods (PDF 287 kb)

Supplementary Data

List of unique peptide identifications (XLS 7892 kb)

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Swaney, D., McAlister, G. & Coon, J. Decision tree–driven tandem mass spectrometry for shotgun proteomics. Nat Methods 5, 959–964 (2008). https://doi.org/10.1038/nmeth.1260

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