Single‐Cell Secretome Profiling Enabled by Selective Enrichment and NanoLC‐MS/MS Analysis

Recent advances in single‐cell proteomics enable the direct profiling of thousands of proteins from a single mammalian cell. However, due to the bottlenecks in detecting low‐abundance secreted proteins and extracellular vesicle (EV) proteins (collectively referred to as the secretome) against a background of high‐abundance proteins in serum‐containing culture medium, the comprehensive investigation of the secretome at the single‐cell level using nanoLC‐MS/MS still remains challenging. Herein, we report a novel single‐cell secretome profiling (SCSP) method by integrating the metabolic labeling of newly synthesized proteins, click chemistry‐based enrichment, and in situ digestion of the labeled secretome in an alkyne‐functionalized capillary micro‐reactor, followed by nanoLC‐MS/MS analysis. By this method, an average of 389 protein groups were quantified from the secretome of single HeLa cells (n = 17), with a total of 752 protein groups confidently identified in the single‐cell secretome, which is a significant increase compared to the previously reported targeted analysis limited to dozens of secreted proteins by antibody recognition. These results indicated that our developed SCSP method would provide a powerful tool to gain insights into secretion heterogeneity and intercellular communication at the single‐cell level.