Improved Detection of Multi-phosphorylated Peptides by LC-MS/MS without Phosphopeptide Enrichment

  • Kim, Suwha (Department of Life Sciences, Gwangju Institute of Science and Technology) ;
  • Choi, Hyunwoo (Department of Life Sciences, Gwangju Institute of Science and Technology) ;
  • Park, Zee-Yong (Department of Life Sciences, Gwangju Institute of Science and Technology)
  • Received : 2007.01.12
  • Accepted : 2007.03.06
  • Published : 2007.06.30


Although considerable effort has been devoted in the mass spectrometric analysis of phosphorylated peptides, successful identification of multi-phosphorylated peptides in enzymatically digested protein samples still remains challenging. The ionization behavior of multi-phosphorylated peptides appears to be somewhat different from that of mono- or di-phosphorylated peptides. In this study, we demonstrate increased sensitivity of detection of multi-phosphorylated peptides of beta casein without using phosphopeptide enrichment techniques. Proteinase K digestion alone increased the detection limit of beta casein multi-phosphorylated peptides in the LC-MS analysis almost 500 fold, compared to conventional trypsin digestion (~50 pmol). In order to understand this effect, various factors affecting the ionization of phosphopeptides were investigated. Unlike ionizations of phosphopeptides with minor modifications, those of multi-phosphorylated peptides appeared to be subject to effects such as selectively suppressed ionization by more ionizable peptides and decreased ionization efficiency by multi-phosphorylation. The enhanced detection limit of multi-phosphorylated peptides resulting from proteinase K digestion was validated using a complex protein sample, namely a lysate of HEK 293 cells. Compared to trypsin digestion, the numbers of phosphopeptides identified and modification sites per peptide were noticeably increased by proteinase K digestion. Non-specific proteases such as proteinase K and elastase have been used in the past to increase detection of phosphorylation sites but the effectiveness of proteinase K digestion for multi-phosphorylated peptides has not been reported.


LC-MS;Multi-Phosphorylated Peptides


  1. Ballif, B. A., Villen, J., Beausoleil, S. A., Schwartz, D., and Gygi, S. P. (2004) Phosphoproteomic analysis of the developing mouse brain. Mol. Cell. Proteomics 3, 1093-1101
  2. Hunter, T. (1995) When is a lipid kinase not a lipid kinase? When it is a protein kinase. Cell 83, 1-4
  3. Li, W., Boykins, R. A., Backlund, P. S., Wang, G., and Chen, H. C. (2002) Identification of phosphoserine and phosphothreonine as cysteic acid and beta-methylcysteic acid residues in peptides by tandem mass spectrometric sequencing. Anal. Chem. 74, 5701-5710
  4. Oda, Y., Nagasu, T., and Chait, B. T. (2001) Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 19, 379-382
  5. Olsen, J. V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., et al. (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635-648
  6. Steen, H., Jebanathirajah, J. A., Rush, J., Morrice, N., and Kirschner, M. W. (2006) Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements. Mol. Cell. Proteomics 5, 172-181
  7. MacCoss, M. J., McDonald, W. H., Saraf, A., Sadygov, R., Clark, J. M., et al. (2002a) Shotgun identification of protein modifications from protein complexes and lens tissue. Proc. Natl. Acad. Sci. USA 99, 7900-7905
  8. Koliakos, G., Trachana, V., Gaitatzi, M., and Dimitriadou, A. (2001) Phosphorylation of laminin-1 by protein kinase C. Mol. Cells 11, 179-185
  9. Kim, J., Camp, D. G., 2nd, and Smith, R. D. (2004) Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry. J. Mass Spectrom. 39, 208-215
  10. McLachlin, D. T. and Chait, B. T. (2001) Analysis of phosphorylated proteins and peptides by mass spectrometry. Curr. Opin. Chem. Biol. 5, 591-602
  11. Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W., and Gygi, S. P. (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. USA 100, 6940-6945
  12. MacCoss, M. J., Wu, C. C., and Yates, J. R., 3rd (2002b) Probability-based validation of protein identifications using a modified SEQUEST algorithm. Anal. Chem. 74, 5593-5599
  13. Liu, S., Zhang, C., Campbell, J. L., Zhang, H., Yeung, K. K., et al. (2005) Formation of phosphopeptide-metal ion complexes in liquid chromatography/electrospray mass spectrometry and their influence on phosphopeptide detection. Rapid Commun. Mass Spectrom. 19, 2747-2756
  14. Annan, R. S., Huddleston, M. J., Verma, R., Deshaies, R. J., and Carr, S. A. (2001) A multidimensional electrospray MS-based approach to phosphopeptide mapping. Anal. Chem. 73, 393-404
  15. Zhou, H., Watts, J. D., and Aebersold, R. (2001) A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375-378
  16. Andersson, L. and Porath, J. (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal. Biochem. 154, 250-254
  17. Knight, Z. A., Schilling, B., Row, R. H., Kenski, D. M., Gibson, B. W., et al. (2003) Phosphospecific proteolysis for mapping sites of protein phosphorylation. Nat. Biotechnol. 21, 1047-1054
  18. Vosseller, K., Hansen, K. C., Chalkley, R. J., Trinidad, J. C., Wells, L., et al. (2005) Quantitative analysis of both protein expression and serine / threo-nine post-translational modifications through stable isotope labeling with dithiothreitol. Proteomics 5, 388-398
  19. Hunter, T. (2000) Signaling--2000 and beyond. Cell 100, 113-127
  20. Mann, M., Ong, S. E., Gronborg, M., Steen, H., Jensen, O. N., et al. (2002) Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol. 20, 261-268
  21. Pandey, A., Podtelejnikov, A. V., Blagoev, B., Bustelo, X. R., Mann, M., et al. (2000) Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc. Natl. Acad. Sci. USA 97, 179-184
  22. Pinkse, M. W., Uitto, P. M., Hilhorst, M. J., Ooms, B., and Heck, A. J. (2004) Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLCESI- MS/MS and titanium oxide precolumns. Anal. Chem. 76, 3935-3943
  23. Schlosser, A., Vanselow, J. T., and Kramer, A. (2005) Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis. Anal. Chem. 77, 5243-5250
  24. Goshe, M. B., Conrads, T. P., Panisko, E. A., Angell, N. H., Veenstra, T. D., et al. (2001) Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses. Anal. Chem. 73, 2578-2586
  25. Beausoleil, S. A., Jedrychowski, M., Schwartz, D., Elias, J. E., Villen, J., et al. (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. USA 101, 12130-12135
  26. Yan, J. X., Packer, N. H., Gooley, A. A., and Williams, K. L. (1998) Protein phosphorylation: technologies for the identification of phosphoamino acids. J. Chromatogr. A 808, 23-41
  27. Blagoev, B., Ong, S. E., Kratchmarova, I., and Mann, M. (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat. Biotechnol. 22, 1139-1145
  28. Garcia, B. A., Shabanowitz, J., and Hunt, D. F. (2005) Analysis of protein phosphorylation by mass spectrometry. Methods 35, 256-264
  29. Zhang, H., Zhang, C., Lajoie, G. A., and Yeung, K. K. (2005) Selective sampling of phosphopeptides for detection by MALDI mass spectrometry. Anal. Chem. 77, 6078-6084
  30. Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., et al. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301-305
  31. McLachlin, D. T. and Chait, B. T. (2003) Improved betaelimination- based affinity purification strategy for enrichment of phosphopeptides. Anal. Chem. 75, 6826-6836
  32. Schlosser, A., Pipkorn, R., Bossemeyer, D., and Lehmann, W. D. (2001) Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spectrometry. Anal. Chem. 73, 170-176