Skip to main content


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


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.


  1. 1.

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

  2. 2.

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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 18.

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

  19. 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.

  20. 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.

  21. 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.

  22. 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.

Download references

Author information

Correspondence to Dongxia Wang or Robert J. Cotter.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation

Key Words

  • Phosphorylation
  • time-of-flight mass spectrometry
  • curved-field reflectron
  • N-terminal sulfonation
  • fragmentation