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

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.

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

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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

Authors

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

Correspondence to Matthias Mann.

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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)

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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

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