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High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics

Nature Biotechnology volume 33, pages 990995 (2015) | Download Citation

Abstract

Mass spectrometry has enabled the study of cellular signaling on a systems-wide scale, through the quantification of post-translational modifications, such as protein phosphorylation1. Here we describe EasyPhos, a scalable phosphoproteomics platform that now allows rapid quantification of hundreds of phosphoproteomes in diverse cells and tissues at a depth of >10,000 sites. We apply this technology to generate time-resolved maps of insulin signaling in the mouse liver. Our results reveal that insulin affects 10% of the liver phosphoproteome and that many known functional phosphorylation sites, and an even larger number of unknown sites, are modified at very early time points (<15 s after insulin delivery). Our kinetic data suggest that the flow of signaling information from the cell surface to the nucleus can occur on very rapid timescales of less than 1 min in vivo. EasyPhos facilitates high-throughput phosphoproteomics studies, which should improve our understanding of dynamic cell signaling networks and how they are regulated and dysregulated in disease.

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Acknowledgements

We thank I. Paron, K. Mayr and G. Sowa for mass spectrometry technical assistance, J. Cox for bioinformatic tools and support, and E.S. Humphrey, M.Y. Hein, N. Kulak, G. Pichler and N. Nagaraj for helpful discussions. S.J.H. is supported by an EMBO (European Molecular Biology Organization) Long Term fellowship, and the project was supported by the Virtual Liver Network (grant 0315748) of the German Federal Ministry of Education and Research (BMBF).

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  1. Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.

    • Sean J Humphrey
    • , S Babak Azimifar
    •  & Matthias Mann

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Contributions

S.J.H. and M.M. conceived the project, interpreted data and wrote the manuscript, S.J.H. developed methods, and performed MS experiments, S.B.A. and S.J.H. designed and performed animal experiments. All authors read and approved the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Matthias Mann.

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https://doi.org/10.1038/nbt.3327

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