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In-Depth Analysis of Protein Phosphorylation by Multidimensional Ion Exchange Chromatography and Mass Spectrometry

  • Maria P. Alcolea
  • Pedro R. Cutillas
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 658)

Abstract

Protein phosphorylation controls fundamental biological functions that are often deregulated in disease. Therefore, system-level understanding of complex pathophysiological processes requires methods that can be used to profile and quantify protein phosphorylation as comprehensively as possible. Here we present a detailed protocol to enrich phosphopeptides from total cell lysates in a form amenable to downstream analysis by mass spectrometry. Using these techniques, we have detected several thousands of phosphorylation sites in the NIH-3T3 cell line.

Key words

Phosphoproteomics cell signaling cancer quantification 

References

  1. 1.
    Manning, G., Whyte, D. B., Martinez, R., Hunter, T., and Sudarsanam, S. (2002) The protein kinase complement of the human genome. Science 298, 1912–1934.PubMedCrossRefGoogle Scholar
  2. 2.
    Bodenmiller, B., Malmstrom, J., Gerrits, B., Campbell, D., Lam, H., Schmidt, A., Rinner, O., Mueller, L. N., Shannon, P. T., Pedrioli, P. G., Panse, C., Lee, H. K., Schlapbach, R., and Aebersold, R. (2007) PhosphoPep―a phosphoproteome resource for systems biology research in Drosophila Kc167 cells. Mol. Syst. Biol. 3, 139.PubMedCrossRefGoogle Scholar
  3. 3.
    de la Fuente van Bentem, S., Mentzen, W. I., de la Fuente, A., and Hirt, H. (2008) Towards functional phosphoproteomics by mapping differential phosphorylation events in signaling networks. Proteomics 8, 4453–4465.CrossRefGoogle Scholar
  4. 4.
    Paradela, A., and Albar, J. P. (2008) Advances in the analysis of protein phosphorylation. J. Proteome Res. 7, 1809–1818.PubMedCrossRefGoogle Scholar
  5. 5.
    Olsen, J. V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P., and Mann, M. (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648.PubMedCrossRefGoogle Scholar
  6. 6.
    Trinidad, J. C., Specht, C. G., Thalhammer, A., Schoepfer, R., and Burlingame, A. L. (2006) Comprehensive identification of phosphorylation sites in postsynaptic density preparations. Mol. Cell. Proteomics 5, 914–922.PubMedCrossRefGoogle Scholar
  7. 7.
    Villen, J., and Gygi, S. P. (2008) The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat. Protoc. 3, 1630–1638.PubMedCrossRefGoogle Scholar
  8. 8.
    Thingholm, T. E., Jensen, O. N., Robinson, P. J., and Larsen, M. R. (2008) SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. Mol. Cell. Proteomics 7, 661–671.PubMedGoogle Scholar
  9. 9.
    Smith, J. C., and Figeys, D. (2008) Recent developments in mass spectrometry-based quantitative phosphoproteomics. Biochem. Cell Biol. 86, 137–148.PubMedCrossRefGoogle Scholar
  10. 10.
    Schreiber, T. B., Mausbacher, N., Breitkopf, S. B., Grundner-Culemann, K., and Daub, H. (2008) Quantitative phosphoproteomics―an emerging key technology in signal-transduction research. Proteomics 8, 4416–4432.PubMedCrossRefGoogle Scholar
  11. 11.
    Andersson, L., and Porath, J. (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal. Biochem. 154, 250–254.PubMedCrossRefGoogle Scholar
  12. 12.
    Tsai, C. F., Wang, Y. T., Chen, Y. R., Lai, C. Y., Lin, P. Y., Pan, K. T., Chen, J. Y., Khoo, K. H., and Chen, Y. J. (2008) Immobilized metal affinity chromatography revisited: pH/acid control toward high selectivity in phosphoproteomics. J. Proteome Res. 7, 4058–4069.PubMedCrossRefGoogle Scholar
  13. 13.
    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-NanoLC–ESI–MS/MS and titanium oxide precolumns. Anal. Chem. 76, 3935–3943.PubMedCrossRefGoogle Scholar
  14. 14.
    Bodenmiller, B., Mueller, L. N., Mueller, M., Domon, B., and Aebersold, R. (2007) Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat. Methods 4, 231–237.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Maria P. Alcolea
    • 1
  • Pedro R. Cutillas
    • 1
  1. 1.Analytical Signalling Group, Centre for Cell SignallingInstitute of Cancer, Bart’s and the London School of Medicine, Queen Mary University of LondonLondonUK

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