Stratifying the Cell Surface Cysteinome using Two-step Enrichment Proteomics

Cell surface cysteines represent an attractive class of residues for chemoproteomic studies due to their accessibility towards drugs and key roles they play in the structure and function of most human proteins. The redox sensitivity of cysteines also makes cell surface cysteines potential markers for cellular activities with altered redox states. However, cell surface cysteines are underrepresented in most of the cysteine proteomic dataset. While many mass spectrometry (MS) based techniques have been developed to enrich membrane proteins, current studies lack methods that can specifically inventory cysteines on cell surfaces. Here, we developed a novel dual enrichment method to achieve chemoproteomic profiling of cell Surface Cysteines - “Cys-Surf''. Combining cell surface capture (CSC) biotinylation and cysteine chemoproteomic biotinylation, this two-step biotinylation platform achieves identification of more than 1,900 cysteines with a specificity of about 50.0% in cell surface localization. In addition, Cys-Surf achieved quantitative analysis of oxidation states of cell surface cysteines, which reflect a completely different profile compared to that of the whole cysteinome. Redox sensitive cell surface cysteines were identified by applying reducing reagents and during T cell activation. Cys-Surf is also compatible with competitive small-molecule screening by isotopic tandem orthogonal activity-based protein profiling (isoTOP-ABPP) to evaluate the ligandability of cell surface cysteines. Altogether, these findings establish a platform that enables redox and ligandability analysis of the cell surface cysteinome and sheds light on future functional studies and drug discovery efforts targeting cell surface cysteines.