Temporal tracking of the insulin action across the cell surface proteome at a resolution of ten seconds

The ligand-receptor signal occurs on cell surface and rapidly triggers a series of events via surface protein alteration. Here, we for the first time report a proteomic platform based on rapid enzymatic surface labeling (PECSL) method and MS-based label-free quantification, to capture the transiently altered proteins in situ and a perturbation-free manner even at a temporal resolution of ten seconds. With this platform, about one thousand GO-annotated cell surface proteins were reproducibly identified in response to insulin stimuli. Notably, we identified thirty-two insulin-regulated proteins on timescales of ten seconds to two minutes, nearly half of which were annotated as insulin/growth factor interactors. In addition to known insulin-regulated proteins of INSR, IGF1R, TFRC, LNPEP, SORT1, novel insulin-regulated proteins from diverse physiological cellular events, like CNNM3, CPD, MRC2, ECE1, were uncovered and further validated. We believe this platform could be a powerful tool to decode the mechanism of many biological processes involving changes of cell surface proteins.