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Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Abstract

Reversible phosphorylation is one of the most important posttranslational modifications of cellular proteins. Mass spectrometry is a widely used technique in the characterization of phosphorylated proteins and peptides. Similar to nonmodified peptides, sequence information for phosphopeptides digested from proteins can be obtained by tandem mass analysis using either electrospray ionization or matrix assisted laser desorption/ionization (MALDI) mass spectrometry. However, the facile loss of neutral phosphoric acid (H3PO4) or HPO3 from precursor ions and fragment ions hampers the precise determination of phosphorylation site, particularly if more than one potential phosphorylation site or concensus sequence is present in a given tryptic peptide. Here, we investigated the fragmentation of phosphorylated peptides under laser-induced dissociation (LID) using a MALDI-time-of-flight mass spectrometer with a curved-field reflectron. Our data demonstrated that intact fragments bearing phosphorylated residues were produced from all tested peptides that contain at least one and up to four phosphorylation sites at serine, threonine, or tyrosine residues. In addition, the LID of phosphopeptides derivatized by N-terminal sulfonation yields simplified MS/MS spectra, suggesting the combination of these two types of spectra could provide an effective approach to the characterization of proteins modified by phosphorylation.

References

  1. Hunter, T. (2000) Signaling: 2000 and beyond.Cell 100, 113–127.

    Article  PubMed  CAS  Google Scholar 

  2. Goffeau, A., Barrell, B. G., Bussey, H., et al. (1996) Life with 6000 genes.Science 274, 546–567.

    Article  PubMed  CAS  Google Scholar 

  3. Annan, R. S. and Carr, S. A. (1996) Phosphopeptide analysis by matrix-assisted laser desorption time-of-flight mass spectrometry.Anal. Chem. 68, 3413–3421.

    Article  PubMed  CAS  Google Scholar 

  4. Busman, M., Schey, K. L., Oatis, J. E., and Knapp, D. R. (1996) Identification of phosphorylation sites in phosphopeptides by positive and negative mode electrospray ionizationtandem mass spectrometry.J. Am. Soc. Mass Spectrom. 7, 243–249.

    Article  CAS  Google Scholar 

  5. DeGnore, J. P. and Qin, J. (1998) Fragmentation of phosphopeptides in an ion trap mass spectrometer.J. Am. Soc. Mass Spectrom. 9, 1175–1188.

    Article  PubMed  CAS  Google Scholar 

  6. Tholey, A., Reed, J., and Lehmann, W. D. (1999) Electrospray tandem mass spectrometric studies of phosphopeptides and phosphopeptide analogues.J. Mass Spectrom. 34, 117–123.

    Article  PubMed  CAS  Google Scholar 

  7. Moyer, S. C., Cotter, R. J., and Woods, A. S. (2002) Fragmentation of phosphopeptides by atmospheric pressure MALDI and ESI/ion trap mass spectrometry.J. Am. Soc. Mass Spectrom. 13, 274–283.

    Article  PubMed  CAS  Google Scholar 

  8. Yu, W., Vath, J. E., Huberty, M. C., and Martin, S. A. (1993) Identification of the facile gasphase cleavage of the Asp-Pro and Asp-Xxx peptide bonds in matrix-assisted laser desorption time-of-flight mass spectrometry.Anal. Chem. 65, 3015–3023.

    Article  PubMed  CAS  Google Scholar 

  9. Wang, D., Thompson, P., Cole, P. A., and Cotter, R. J. (2005) Structural analysis of a highly acetylated protein using a curved-field reflectron mass spectrometer.Proteomics 5, 2288–2296.

    Article  PubMed  CAS  Google Scholar 

  10. Håkansson, K., Chalmers, M. J., Quinn, J. P., McFarland, M. A., Hendrickson, C. L., and Marshall, A. G. (2003) Combined electron capture and infrared multiphoton dissociation for multistage MS/MS in a fourier transform ion cyclotron resonance mass spectrometer.Anal. Chem. 75, 3256–3262.

    Article  PubMed  CAS  Google Scholar 

  11. Zubarev, R. A., Kelleher, N. L., and McLafferty, F. W. (1998) Electron capture dissociation of multiply charged protein cations. A non-ergodic process.J. Am. Chem. Soc. 120, 3265–3266.

    Article  CAS  Google Scholar 

  12. Leymarie, N., Costello, C. E., O’Connor, P. B. (2003) Electron capture dissociation initiates a free radical reaction cascade.J. Am. Chem. Soc. 125, 8949–8958.

    Article  PubMed  CAS  Google Scholar 

  13. Stenballe, A., Jensen, O. N., Olsen, J. V., Haselmann, K. F., and Zubarev, R. A. (2000) Electron capture dissociation of singly and multiply phosphorylated peptides.Rapid Commun. Mass Spectrom. 14, 1793–1800.

    Article  Google Scholar 

  14. Shi, S. D.-H., Hemling, M. E., Carr, S. A., Horn, D. A., Lindh, I., and McLafferty, F. W. (2001) Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry.Anal. Chem. 73, 19–22.

    Article  PubMed  CAS  Google Scholar 

  15. Cooper, H. J., Hakansson, K., and Marshall, A. G. (2005) The role of electron capture dissociation in biomolecular analysis.Mass Spectrometry Reviews 24, 201–222.

    Article  PubMed  CAS  Google Scholar 

  16. Taylor, G. K., Kim, Y. B., Forbes, A. J., Meng, F., McCarthy, R., and Kelleher, N. L. (2003) Web and database software for identification of intact proteins using “top down” mass spectrometry.Anal. Chem. 75, 4081–4086.

    Article  PubMed  CAS  Google Scholar 

  17. Rosario, M., Domingues, M., Marques, G. O. S., et al. (1999) Do charge-remote fragmentations occur under matrix-assisted laser desorption ionization post-source decompositions and matrix-assisted laser desorption collisionally activated decompositions?J. Amer. Soc. Mass Spectrom. 10, 217–223.

    Article  Google Scholar 

  18. Cheng, C., Pittenauer, E., and Gross, M. (1998) Charge-remote fragmentations are energy-dependent processes.J. Amer. Soc. Mass Spectrom. 9, 840–844.

    Article  CAS  Google Scholar 

  19. Kaufmann, R., Spengler, B., and Lutzenkirchen, F. (1993) Mass spectrometric sequencing of linear peptides by product-ion analysis in a reflectron time-of-flight mass spectrometer using matrix-assisted laser desorption ionization.Rapid Commun. Mass Spectrom. 7, 902–910.

    Article  PubMed  CAS  Google Scholar 

  20. Cotter, R. J., Gardner, B., Iltchenko, S., and English, R. D. (2004) Tandem time-of-flight mass spectrometry with a curved field reflectron.Anal. Chem. 76, 1976–1981.

    Article  PubMed  CAS  Google Scholar 

  21. Wang, D., Kalb, S. R., and Cotter, R. J. (2004) Improved procedures for N-terminal sulfonation of peptides for MALDI PSD peptide sequencing.Rapid. Commun. Mass Spectrom. 18, 96–102.

    Article  PubMed  CAS  Google Scholar 

  22. Keough, T., Youngquist, R. S., and Lacey, M. P. (2003) Sulfonic acid derivatives for peptide sequencing by MALDI MS.Anal. Chem. 75, 157A-165A.

    Article  Google Scholar 

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Correspondence to Robert J. Cotter.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Wang, D., Cole, P.A. & Cotter, R.J. Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Clin Proteom 2, 133–144 (2006). https://doi.org/10.1007/BF02752496

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