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.

Reproducible isolation of distinct, overlapping segments of the phosphoproteome

Abstract

The ability to routinely analyze and quantitatively measure changes in protein phosphorylation on a proteome-wide scale is essential for biological and clinical research. We assessed the ability of three common phosphopeptide isolation methods (phosphoramidate chemistry (PAC), immobilized metal affinity chromatography (IMAC) and titanium dioxide) to reproducibly, specifically and comprehensively isolate phosphopeptides from complex mixtures. Phosphopeptides were isolated from aliquots of a tryptic digest of the cytosolic fraction of Drosophila melanogaster Kc167 cells and analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry. Each method reproducibly isolated phosphopeptides. The methods, however, differed in their specificity of isolation and, notably, in the set of phosphopeptides isolated. The results suggest that the three methods detect different, partially overlapping segments of the phosphoproteome and that, at present, no single method is sufficient for a comprehensive phosphoproteome analysis.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Strategy used to analyze the reproducibility, overlap and specificity of the different phosphopeptide isolation methods.
Figure 2: Similarity and overlap within and between isolates of the investigated phosphopeptide isolation methods.
Figure 3: Comparison of LC-MS base peak chromatograms for isolation products from the investigated methods.
Figure 4: Overlap between phosphopeptide isolation methods on the level of identified phosphorylation sites.
Figure 5: Characteristics of phosphopeptides identified from PAC, IMAC and TiO2 isolates.

References

  1. Garavelli, J.S. The RESID Database of Protein Modifications as a resource and annotation tool. Proteomics 4, 1527–1533 (2004).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  3. Aebersold, R. & Goodlett, D.R. Mass spectrometry in proteomics. Chem. Rev. 101, 269–295 (2001).

    CAS  Article  Google Scholar 

  4. Sachon, E., Mohammed, S., Bache, N. & Jensen, O.N. Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis. Rapid Commun. Mass Sp. 20, 1127–1134 (2006).

    CAS  Article  Google Scholar 

  5. Goshe, M.B. et al. Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses. Anal. Chem. 73, 2578–2586 (2001).

    CAS  Article  Google Scholar 

  6. Blagoev, B., Ong, S.E., Kratchmarova, I. & Mann, M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat. Biotechnol. 22, 1139–1145 (2004).

    CAS  Article  Google Scholar 

  7. Gruhler, A. et al. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Mol. Cell. Proteomics 4, 310–327 (2005).

    CAS  Article  Google Scholar 

  8. Tao, W.A. et al. Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. Nat. Methods 2, 591–598 (2005).

    CAS  Article  Google Scholar 

  9. Zhou, H., Watts, J.D. & Aebersold, R. A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375–378 (2001).

    CAS  Article  Google Scholar 

  10. Reinders, J. & Sickmann, A. State-of-the-art in phosphoproteomics. Proteomics 5, 4052–4061 (2005).

    CAS  Article  Google Scholar 

  11. Gold, M.R. et al. Purification and identification of tyrosine-phosphorylated proteins from B lymphocytes stimulated through the antigen receptor. Electrophoresis 15, 441–453 (1994).

    CAS  Article  Google Scholar 

  12. Kanakura, Y., Druker, B., DiCarlo, J., Cannistra, S.A. & Griffin, J.D. Phorbol 12-myristate 13-acetate inhibits granulocyte-macrophage colony stimulating factor-induced protein tyrosine phosphorylation in a human factor-dependent hematopoietic cell line. J. Biol. Chem. 266, 490–495 (1991).

    CAS  PubMed  Google Scholar 

  13. Andersson, L. & Porath, J. Isolation of phosphoproteins by immobilized metal (Fe-3+) affinity-chromatography. Anal. Biochem. 154, 250–254 (1986).

    CAS  Article  Google Scholar 

  14. Rush, J. et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 23, 94–101 (2005).

    CAS  Article  Google Scholar 

  15. Li, S. & Dass, C. Iron(III)-immobilized metal ion affinity chromatography and mass spectrometry for the purification and characterization of synthetic phosphopeptides. Anal. Biochem. 270, 9–14 (1999).

    CAS  Article  Google Scholar 

  16. Knight, Z.A. et al. Phosphospecific proteolysis for mapping sites of protein phosphorylation. Nat. Biotechnol. 21, 1047–1054 (2003).

    CAS  Article  Google Scholar 

  17. Oda, Y., Nagasu, T. & Chait, B.T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 19, 379–382 (2001).

    CAS  Article  Google Scholar 

  18. Bodenmiller, B. et al. An integrated chemical, mass spectrometric and computational strategy for phosphoproteome analysis: application to human jurkat T cells. Mol. Biosyst. (in the press).

  19. Posewitz, M.C. & Tempst, P. Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal. Chem. 71, 2883–2892 (1999).

    CAS  Article  Google Scholar 

  20. Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P. & Jorgensen, T.J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 4, 873–886 (2005).

    CAS  Article  Google Scholar 

  21. Pinkse, M.W.H., Uitto, P.M., Hilhorst, M.J., Ooms, B. & Heck, A.J.R. 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 (2004).

    CAS  Article  Google Scholar 

  22. Kweon, H.K. & Hakansson, K. Selective zirconium dioxide-based enrichment of phosphorylated peptides for mass spectrometric analysis. Anal. Chem. 78, 1743–1749 (2006).

    CAS  Article  Google Scholar 

  23. Cao, P. & Stults, J.T. Phosphopeptide analysis by on-line immobilized metal-ion affinity chromatography-capillary electrophoresis-electrospray ionization mass spectrometry. J. Chromatogr. A. 853, 225–235 (1999).

    CAS  Article  Google Scholar 

  24. Ficarro, S.B. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 (2002).

    CAS  Article  Google Scholar 

  25. Nuhse, T.S., Stensballe, A., Jensen, O.N. & Peck, S.C. Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry. Mol. Cell. Proteomics 2, 1234–1243 (2003).

    Article  Google Scholar 

  26. Elias, J.E., Haas, W., Faherty, B.K. & Gygi, S.P. Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations. Nat. Methods 2, 667–675 (2005).

    CAS  Article  Google Scholar 

  27. Mann, M. et al. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol. 20, 261–268 (2002).

    CAS  Article  Google Scholar 

  28. Schwartz, D. & Gygi, S.P. An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat. Biotechnol. 23, 1391–1398 (2005).

    CAS  Article  Google Scholar 

  29. Schlosser, A., Pipkorn, R., Bossemeyer, D. & Lehmann, W.D. Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spectrometry. Anal. Chem. 73, 170–176 (2001).

    CAS  Article  Google Scholar 

  30. Larsen, M.R., Graham, M.E., Robinson, P.J. & Roepstorff, P. Improved detection of hydrophilic phosphopeptides using graphite powder microcolumns and mass spectrometry: evidence for in vivo doubly phosphorylated dynamin I and dynamin III. Mol. Cell. Proteomics 3, 456–465 (2004).

    CAS  Article  Google Scholar 

  31. Keller, A., Nesvizhskii, A.I., Kolker, E. & Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383–5392 (2002).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank O. Rinner, J. Malmström, P. Picotti and M. Larsen for fruitful discussions. This project has been funded in part by ETH Zurich and by federal funds from the US National Heart, Lung, and Blood Institute of the National Institutes of Health (N01-HV-28179). B.B. is the recipient of a fellowship by the Boehringer Ingelheim Fonds.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ruedi Aebersold.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Amino acid usage of the identified (phospho)peptides compared to the starting material and the used protein database. (PDF 88 kb)

Supplementary Fig. 2

The influence of the peptide to resin ratio on the selectivity and percentage of singly phosphorylated peptides in the case of pTiO2. (PDF 66 kb)

Supplementary Fig. 3

The influence of the peptide to resin ratio on the selectivity and percentage of singly phosphorylated peptides in the case of IMAC. (PDF 21 kb)

Supplementary Table 1

Values of the similarity and overlap between any two LC-MS runs as computed by Superhirn. (PDF 99 kb)

Supplementary Table 2

Phosphopeptides identified with PAC, IMAC, pTiO2 and dhbTiO2. (PDF 214 kb)

Supplementary Table 3

Influence of methylation prior and/or after phosphopeptide isolation using IMAC. (PDF 156 kb)

Supplementary Methods (PDF 93 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bodenmiller, B., Mueller, L., Mueller, M. et al. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods 4, 231–237 (2007). https://doi.org/10.1038/nmeth1005

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth1005

Further reading

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