Ultrafast and Chemoselective Biotinylation of Living Cell Surfaces for Time-Resolved Surfaceome Analysis

Cell surface proteins participate in many important biological processes, such as cell-to-cell interaction, signal transduction, cell adhesion, and protein transportation. In-depth study of the cell surface protein group is of great significance. Nevertheless, detection and analysis of the surfaceome remain a significant challenge due to their low abundance and hydrophobicity. Herein, we reported an ultrafast and chemoselective labeling method using our newly developed trifunctional probe, the OPA-S-S-alkyne, which labeled cell surface lysine residues, and then established a novel cell surfaceome profiling approach. According to our experimental results, the OPA-S-S-alkyne probe can react extremely fast with living cells, labeling cells in only 1 min, while traditional NHS (labeling cell surface lysine with N-hydroxysuccinimide ester probe) and CSC (labeling cell surface glycan with hydrazide biotin probe) methods normally take longer time of more than 30 min and 1 h, respectively. Taking advantage of this ultrafast property of the method, we highlight the utility of this method by exploring the temporal dynamic changes of surfaceome upon EGF stimulation in living Hela cells and reported "early" and "late" EGF-regulated cell surface proteins, which are difficult to be distinguished by the current cell surface profiling approaches.