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A systematic approach to the analysis of protein phosphorylation

Nature Biotechnology volume 19, pages 375–378 (2001)Cite this article

Abstract

Reversible protein phosphorylation has been known for some time to control a wide range of biological functions and activities1,2,3. Thus determination of the site(s) of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. However, direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date, typically requiring the purification to homogeneity of the phosphoprotein of interest before analysis4,5,6. Thus, there has been a substantial need for a more rapid and general method for the analysis of protein phosphorylation in complex protein mixtures. Here we describe such an approach to protein phosphorylation analysis. It consists of three steps: (1) selective phosphopeptide isolation from a peptide mixture via a sequence of chemical reactions, (2) phosphopeptide analysis by automated liquid chromatography–tandem mass spectrometry (LC-MS/MS), and (3) identification of the phosphoprotein and the phosphorylated residue(s) by correlation of tandem mass spectrometric data with sequence databases. By utilizing various phosphoprotein standards and a whole yeast cell lysate, we demonstrate that the method is equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins, and is capable of selectively isolating and identifying phosphopeptides present in a highly complex peptide mixture.

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Figure 1: Phosphopeptide isolation strategy and validation with phosphoprotein β-casein.
Figure 2: Phosphopeptide isolation from LCK–MBP kinase reaction mixture.
Figure 3: Phosphopeptide isolation from yeast cell lysate.

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References

  1. Graves, J.D. & Krebs, E.D. Protein phosphorylation and signal transduction. Pharmacol. Ther. 82, 111–121 (1999).

    Article  CAS  Google Scholar 

  2. Koch, C.A., Anderson, D., Moran, M. F., Ellis, C. & Pawson, T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science 252, 668–674 (1991).

    Article  CAS  Google Scholar 

  3. Hunter, T. 1001 protein kinases redux—towards 2000. Semin. Cell Biol. 5, 367–376 (1994).

    Article  CAS  Google Scholar 

  4. Verma, R. et al. Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase. Science 278, 455–460 (1997).

    Article  CAS  Google Scholar 

  5. Watts, J.D. et al. Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70. J. Biol. Chem. 269, 29520–29529 (1994).

    CAS  PubMed  Google Scholar 

  6. Gingras, A.C. et al. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev. 13, 1422–1437 (1999).

    Article  CAS  Google Scholar 

  7. Bodanszky, A.B.M. (ed.) The practice of peptide synthesis, Vol. 21. (Springer-Verlag, New York; 1984).

    Book  Google Scholar 

  8. Hoare, D.G. & Koshland, D.E. Jr. A method for the quantitative modification and estimation of carboxylic acid groups in proteins. J. Biol. Chem. 242, 2447–2453 (1967).

    CAS  PubMed  Google Scholar 

  9. Chu, B., Wahl, G.M. & Orgel, L.E. Derivatization of unprotected polynucleotides. Nucleic Acids Res. 11, 6513–6529 (1983).

    Article  CAS  Google Scholar 

  10. Papayannopoulos, I.A. The interpretation of collision-induced dissociation tandem mass spectra of peptides. Mass Spectrom. Rev. 14, 49–73 (1995).

    Article  CAS  Google Scholar 

  11. Jonscher, K.R. & Yates, J.R. Matrix-assisted laser desorption ionization/quadrupole ion trap mass spectrometry of peptides. Application to the localization of phosphorylation sites on the P protein from Sendai virus. J. Biol. Chem. 272, 1735–1741 (1997).

    Article  CAS  Google Scholar 

  12. Qin, J. & Chait, B.T. Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry. Anal. Chem. 69, 4002–4009 (1997).

    Article  CAS  Google Scholar 

  13. Aebersold, R., Watts, J.D., Morrison, H.D. & Bures, E.J. Determination of the site of tyrosine phosphorylation at the low picomole level by automated solid-phase sequence analysis. Anal. Biochem. 199, 51–60. (1991).

    Article  CAS  Google Scholar 

  14. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    Article  CAS  Google Scholar 

  15. Gygi, S.P., Rochon, Y., Franza, B.R. & Aebersold, R. Correlation between protein and mRNA abundance in yeast. Mol. Cell. Biol. 19, 1720–1730 (1999).

    Article  CAS  Google Scholar 

  16. Futcher, B., Latter, G.I., Monardo, P., McLaughlin, C.S., Garrels, J.I. A sampling of the yeast proteome. Mol. Cell. Biol. 19, 7357–7368 (1999).

    Article  CAS  Google Scholar 

  17. Moore, D.D. et al. (eds). Current protocols in molecular biology. (Wiley, New York; 1987).

    Google Scholar 

  18. Carraway, K.L. & Koshland, D.E. Jr. Reaction of tyrosine residues in proteins with carbodiimide reagents. Biochim. Biophys. Acta 160, 272–274 (1968).

    Article  CAS  Google Scholar 

  19. Eng, J., McCormack, A.L. & Yates, J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

The National Science Foundation Science and Technology Center for Molecular Biotechnology, the National Institutes of Health (RO1 A141109 and 1R33 CA84698), the NIH Research Resource Center (RR11823), and the Merck Genome Research Institute provided support for this work. We thank Drs. Beate Rist, Steven P. Gygi, and David R. Goodlett for helpful discussions.

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  1. Huilin Zhou, Julian D. Watts & Ruedi Aebersold

    Present address: Institute for Systems Biology, 4225 Roosevelt Way NE, Suite 200, Seattle, WA, 98105

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  1. Department of Molecular Biotechnology, University of Washington, Seattle, 98195, WA

    Huilin Zhou, Julian D. Watts & Ruedi Aebersold

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Correspondence to Ruedi Aebersold.

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Zhou, H., Watts, J. & Aebersold, R. A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol 19, 375–378 (2001). https://doi.org/10.1038/86777

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