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(1). 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 similar to 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.