Skip to main content

Thank you for visiting 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.

Large-scale phosphosite quantification in tissues by a spike-in SILAC method


Despite progress in mass spectrometry (MS)-based phosphoproteomics, large-scale in vivo analyses remain challenging. Here we report a 'spike-in' stable-isotope labeling with amino acids in cell culture (SILAC) methodology using standards derived from labeled mouse liver cell lines, using which we analyzed insulin signaling. With this approach we identified 15,000 phosphosites and quantitatively compared 10,000 sites in response to insulin treatment, creating a very large, accurately quantified in vivo phosphoproteome dataset.

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.


All prices are NET prices.

Figure 1: Liver quantitative phosphoproteomic analyses.
Figure 2: Insulin signaling pathway members activated in liver upon insulin treatment.


  1. Witze, E.S., Old, W.M., Resing, K.A. & Ahn, N.G. Nat. Methods 4, 798–806 (2007).

    CAS  Article  Google Scholar 

  2. Bodenmiller, B., Mueller, L.N., Mueller, M., Domon, B. & Aebersold, R. Nat. Methods 4, 231–237 (2007).

    CAS  Article  Google Scholar 

  3. Boersema, P.J., Mohammed, S. & Heck, A.J. J. Mass Spectrom. 44, 861–878 (2009).

    CAS  Article  Google Scholar 

  4. Choudhary, C. & Mann, M. Nat. Rev. Mol. Cell Biol. 11, 427–439 (2010).

    CAS  Article  Google Scholar 

  5. Huttlin, E.L. et al. Cell 143, 1174–1189 (2010).

    CAS  Article  Google Scholar 

  6. Villen, J., Beausoleil, S.A., Gerber, S.A. & Gygi, S.P. Proc. Natl. Acad. Sci. USA 104, 1488–1493 (2007).

    CAS  Article  Google Scholar 

  7. Wisniewski, J.R., Nagaraj, N., Zougman, A., Gnad, F. & Mann, M. J. Proteome Res. 9, 3280–3289 (2010).

    CAS  Article  Google Scholar 

  8. Kruger, M. et al. Cell 134, 353–364 (2008).

    Article  Google Scholar 

  9. Olsen, J.V. et al. Cell 127, 635–648 (2006).

    CAS  Article  Google Scholar 

  10. Geiger, T., Cox, J., Ostasiewicz, P., Wisniewski, J.R. & Mann, M. Nat. Methods 7, 383–385 (2010).

    CAS  Article  Google Scholar 

  11. Taniguchi, C.M., Emanuelli, B. & Kahn, C.R. Nat. Rev. Mol. Cell Biol. 7, 85–96 (2006).

    CAS  Article  Google Scholar 

  12. Geiger, T. et al. Nat. Protoc. 6, 147–157 (2011).

    CAS  Article  Google Scholar 

  13. Ishihama, Y. et al. Nat. Biotechnol. 23, 617–621 (2005).

    CAS  Article  Google Scholar 

  14. Wisniewski, J.R., Zougman, A., Nagaraj, N. & Mann, M. Nat. Methods 6, 359–362 (2009).

    CAS  Article  Google Scholar 

  15. Olsen, J.V. et al. Mol. Cell. Proteomics 8, 2759–2769 (2009).

    CAS  Article  Google Scholar 

  16. Olsen, J.V. et al. Nat. Methods 4, 709–712 (2007).

    CAS  Article  Google Scholar 

  17. Nagaraj, N., D'Souza, R.C., Cox, J., Olsen, J.V. & Mann, M. J. Proteome Res. 9, 6786–6794 (2010).

    CAS  Article  Google Scholar 

  18. Cox, J. & Mann, M. Nat. Biotechnol. 26, 1367–1372 (2008).

    CAS  Article  Google Scholar 

  19. Cox, J. et al. J. Proteome Res. 10, 1794–1805 (2011).

    CAS  Article  Google Scholar 

Download references


We thank the members of our laboratory for fruitful discussions and help. T. Walther, R. Farese, B. Blagoev and I. Kratchmarova critically read the manuscript. This project was supported by HepatoSys and by Diabetes Genome Anatomy Project (US National Institutes of Health grant DK60837).

Author information

Authors and Affiliations



M. Monetti designed and performed the experiments, analyzed the data and wrote the paper. N.N. contributed to experiments, data analysis and writing the manuscript. K.S. assisted with data analysis and writing the manuscript. M. Mann supervised the work and wrote the paper.

Corresponding author

Correspondence to Matthias Mann.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Table 1 and Supplementary Note (PDF 2084 kb)

Supplementary Table 2

List of all class I sites quantified in at least two experiments with details including the coefficient of variation and directional variability of the ratios. (XLS 14899 kb)

Supplementary Table 3

List of regulated class I sites and sites that are exclusively quantified either in insulin- or PBS- treated samples. (XLS 2075 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Monetti, M., Nagaraj, N., Sharma, K. et al. Large-scale phosphosite quantification in tissues by a spike-in SILAC method. Nat Methods 8, 655–658 (2011).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


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