High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics.
Mass spectrometry has enabled the study of cellular signaling on a systems-wide scale, through the quantification of post-translational modifications, such as protein phosphorylation. 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.
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
Sean J
SJ
Humphrey
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
S Babak
SB
Azimifar
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
Matthias
M
Mann
D000818:Animals; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D007328:Insulin / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D010750:Phosphoproteins / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods; D020543:Proteome / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D007328:Insulin; D010750:Phosphoproteins; D020543:Proteome