Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome

Nature Biotechnology volume 19, pages 379–382 (2001)Cite this article

Abstract

The current progression from genomics to proteomics is fueled by the realization that many properties of proteins (e.g., interactions, post-translational modifications) cannot be predicted from DNA sequence1. Although it has become feasible to rapidly identify proteins from crude cell extracts using mass spectrometry after two-dimensional electrophoretic separation, it can be difficult to elucidate low-abundance proteins of interest in the presence of a large excess of relatively abundant proteins2,3. Therefore, for effective proteome analysis it becomes critical to enrich the sample to be analyzed in subfractions of interest. For example, the analysis of protein kinase substrates can be greatly enhanced by enriching the sample of phosphorylated proteins. Although enrichment of phosphotyrosine-containing proteins has been achieved through the use of high-affinity anti-phosphotyrosine antibodies4, the enrichment of phosphoserine/threonine-containing proteins has not been routinely possible. Here, we describe a method for enriching phosphoserine/threonine-containing proteins from crude cell extracts, and for subsequently identifying the phosphoproteins and sites of phosphorylation. The method, which involves chemical replacement of the phosphate moieties by affinity tags, should be of widespread utility for defining signaling pathways and control mechanisms that involve phosphorylation or dephosphorylation of serine/threonine residues.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: (A) Scheme for isolating/enriching phosphoproteins and/or phosphopeptides.
Figure 2: Enrichment and isolation of a phosphoprotein from a complex mixture of proteins.
Figure 3: MALDI mass spectra of a tryptic digest of a whole-cell yeast (Saccharomyces cerevisiae) extract to which 2% by weight of ovalbumin was added.
Figure 4: ESI-MS/MS spectra of biotinylated proteins allow identification of the formerly phosphorylated proteins and identification of the sites of biotinylation (i.e., phosphorylation).

Similar content being viewed by others

References

  1. Lander, E.S. The new genomics: global views of biology. Science 274, 536–539 (1996).

    Article  CAS  Google Scholar 

  2. Gygi, S.P., Corthals, G.L., Zhang, Y., Rochon, Y. & Aebersold, R. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc. Natl. Acad. Sci. USA 97, 9390–9395 (2000).

    Article  CAS  Google Scholar 

  3. Corthals, G.L., Wasinger, V.C., Hochstrasser, D.F. & Sanchez, J.C. The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis 21, 1104–1115 (2000).

    Article  CAS  Google Scholar 

  4. Pandey, A. et al. 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 (2000).

    Article  CAS  Google Scholar 

  5. Taborsky, G. Phosphoproteins. Adv. Protein. Chem. 28, 1–210 (1974).

    Article  CAS  Google Scholar 

  6. Annan, W.D., Manson, W. & Nimmo, J.A. The identification of phosphoseryl residues during the determination amino acid sequence in phosphoproteins. Anal. Biochem. 121, 62–68 (1982).

    Article  CAS  Google Scholar 

  7. Meyer, H.E., Hoffmann-Posorske, E., Korte, H. & Heilmeyer, L.M., Jr. Sequence analysis of phosphoserine-containing peptides. Modification for picomolar sensitivity. FEBS Lett. 204, 61–66 (1986).

    Article  CAS  Google Scholar 

  8. Holmes, C.F. A new method for the selective isolation of phosphoserine-containing peptides. FEBS Lett. 215, 21–24 (1987).

    Article  CAS  Google Scholar 

  9. Mega, T., Nakamura, N. & Ikenaka, T. Modifications of substituted seryl and threonyl residues in phosphopeptides and a polysialoglycoprotein by beta-elimination and nucleophile additions. J. Biochem. (Tokyo) 107, 68–72 (1990).

    Article  CAS  Google Scholar 

  10. Meyer, H.E., Hoffmann-Posorske, E. & Heilmeyer, L.M., Jr. Determination and location of phosphoserine in proteins and peptides by conversion to S-ethylcysteine. Methods Enzymol. 201, 169–185 (1991).

    Article  CAS  Google Scholar 

  11. Meyer, H.E. et al. Strategies for nonradioactive methods in the localization of phosphorylated amino acids in proteins. FASEB J. 7, 776–782 (1993).

    Article  CAS  Google Scholar 

  12. Moorhead, G., MacKintosh, R.W., Morrice, N., Gallagher, T. & MacKintosh, C. Purification of type 1 protein (serine/threonine) phosphatases by microcystin–Sepharose affinity chromatography. FEBS Lett. 356, 46–50 (1994).

    Article  CAS  Google Scholar 

  13. Fadden, P. & Haystead, T.A. Quantitative and selective fluorophore labeling of phosphoserine on peptides and proteins: characterization at the attomole level by capillary electrophoresis and laser-induced fluorescence. Anal. Biochem. 225, 81–88 (1995).

    Article  CAS  Google Scholar 

  14. Jaffe, H., Veeranna & Pant, H.C. Characterization of serine and threonine phosphorylation sites in beta-elimination/ethanethiol addition-modified proteins by electrospray tandem mass spectrometry and database searching. Biochemistry 37, 16211–16224 (1998).

    Article  CAS  Google Scholar 

  15. Patchornik, A. & Sokolovsky, M. Nonenzymatic cleavages of peptide chains at the cysteine and serine residues through their conversion into dehydroalanine. I. Hydrolytic and oxidative cleavage of dehydroalanine residues. J. Am. Chem. Soc. 86, 1206–1212 (1964).

    Article  CAS  Google Scholar 

  16. Zhang, W. & Chait, B.T. ProFound: an expert system for protein identification using mass spectrometric peptide mapping information. Anal. Chem. 72, 2482–2489 (2000).

    Article  CAS  Google Scholar 

  17. Fenyo, D., Qin, J. & Chait, B.T. Protein identification using mass spectrometric information. Electrophoresis 19, 998–1005 (1998).

    Article  CAS  Google Scholar 

  18. Sechi, S. & Chait, B.T. Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification. Anal. Chem. 70, 5150–5158 (1998).

    Article  CAS  Google Scholar 

  19. Biemann, K. Contributions of mass spectrometry to peptide and protein structure. Biomed. Environ. Mass Spectrom. 16, 99–111 (1988).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Center for Research Resources, National Institutes of Health (Grant RR00862 to B.T.C.), and the Eisai Co., Ltd. We thank Fred Cross, Angus Nairn, Derek McLachlin, Yasutaka Takase, and Takashi Owa for useful discussions, Wenzhu Zhang and David Fenyö for development of the protein identification software, and Kappei Tsukahara for assistance in yeast culture and cell handling.

Author information

Authors and Affiliations

  1. The Rockefeller University, 1230 York Avenue, New York, 10021, NY, USA

    Yoshiya Oda & Brian T. Chait

  2. Eisai Co., Ltd., Tokodai 5-1-3, Tsukuba, 300-2635, Ibaraki, Japan

    Yoshiya Oda & Takeshi Nagasu

Authors
  1. Yoshiya Oda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takeshi Nagasu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Brian T. Chait
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Brian T. Chait.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Oda, Y., Nagasu, T. & Chait, B. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat Biotechnol 19, 379–382 (2001). https://doi.org/10.1038/86783

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/86783

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing