Temporal Tracking of Insulin Action on the Cell Surface of Proteins at a Resolution of Ten Seconds

Abstract

The ligand–receptor signaling occurring on the cell surface governs cell growth, proliferation, and survival via rapidly triggering a cascade of events. Here, we for the first time report an in situ perturbation-free and rapid surface proteomic profiling at a temporal resolution of ten seconds. By this innovation, about 1022 cell surface-associated proteins were reproducibly identified and quantified. It is noteworthy that, upon a model ligand insulin stimulus, a few rapid-responding proteins at 10 s to 2 min were identified, e.g., CNNM3. Moreover, temporal response patterns were established for the members of GLUT4 storage vesicles (GSVs; responsible for glucose transportation) and confirmed with five known GSV proteins. This pattern was then exploited to uncover seven new regulatory proteins (LDLR, HFE, ECE1, MRC2, CORO1C, CPD, and BST2). Collectively, we showed a powerful surface proteomic tool to decipher rapid signaling of cell-surface proteins and to uncover new subunits involved in rapidly trafficking vesicles.

Painting cell-cell interactions by horseradish peroxidase and endogenously generated hydrogen peroxide

“…This reaction enables protein labeling by forming covalent bonds between biotin-phenoxy radicals and electron-rich amino acid residues, such as tyrosine. Previous studies have utilized exogenous HRP, split HRP, or nucleic acid-based HRP mimics, such as G-quadruplex/hemin, to label and identify surface proteomes and intercellular protein-protein interactions in various in vitro or ex vivo models [20, 21, 22, 23] . However, the toxicity of exogenous H 2 O 2 treatment limits its application.…”