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.