Abstract
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.
Similar content being viewed by others
References
Witze, E.S., Old, W.M., Resing, K.A. & Ahn, N.G. Nat. Methods 4, 798–806 (2007).
Bodenmiller, B., Mueller, L.N., Mueller, M., Domon, B. & Aebersold, R. Nat. Methods 4, 231–237 (2007).
Boersema, P.J., Mohammed, S. & Heck, A.J. J. Mass Spectrom. 44, 861–878 (2009).
Choudhary, C. & Mann, M. Nat. Rev. Mol. Cell Biol. 11, 427–439 (2010).
Huttlin, E.L. et al. Cell 143, 1174–1189 (2010).
Villen, J., Beausoleil, S.A., Gerber, S.A. & Gygi, S.P. Proc. Natl. Acad. Sci. USA 104, 1488–1493 (2007).
Wisniewski, J.R., Nagaraj, N., Zougman, A., Gnad, F. & Mann, M. J. Proteome Res. 9, 3280–3289 (2010).
Kruger, M. et al. Cell 134, 353–364 (2008).
Olsen, J.V. et al. Cell 127, 635–648 (2006).
Geiger, T., Cox, J., Ostasiewicz, P., Wisniewski, J.R. & Mann, M. Nat. Methods 7, 383–385 (2010).
Taniguchi, C.M., Emanuelli, B. & Kahn, C.R. Nat. Rev. Mol. Cell Biol. 7, 85–96 (2006).
Geiger, T. et al. Nat. Protoc. 6, 147–157 (2011).
Ishihama, Y. et al. Nat. Biotechnol. 23, 617–621 (2005).
Wisniewski, J.R., Zougman, A., Nagaraj, N. & Mann, M. Nat. Methods 6, 359–362 (2009).
Olsen, J.V. et al. Mol. Cell. Proteomics 8, 2759–2769 (2009).
Olsen, J.V. et al. Nat. Methods 4, 709–712 (2007).
Nagaraj, N., D'Souza, R.C., Cox, J., Olsen, J.V. & Mann, M. J. Proteome Res. 9, 6786–6794 (2010).
Cox, J. & Mann, M. Nat. Biotechnol. 26, 1367–1372 (2008).
Cox, J. et al. J. Proteome Res. 10, 1794–1805 (2011).
Acknowledgements
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
Contributions
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
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
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). https://doi.org/10.1038/nmeth.1647
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth.1647
- Springer Nature America, Inc.
This article is cited by
-
Cell-type-specific metabolic labeling of nascent proteomes in vivo
Nature Biotechnology (2017)
-
Fatty acid synthesis configures the plasma membrane for inflammation in diabetes
Nature (2016)
-
Identification of KasA as the cellular target of an anti-tubercular scaffold
Nature Communications (2016)
-
High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics
Nature Biotechnology (2015)
-
Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion
Nature Communications (2015)