Cell surface-exposed proteins (CSPs), termed the surfaceome, play a key role in many cellular processes.In-depth CSP analysis is significant for screening candidate biomarkers and drug targets. Highly selective enrichment of CSPs in physiological cellular environments is attractive but remains technically challenging.Here, we present a photocrosslinking-assisted cell surface protein enrichment (PCSPE) strategy.In this strategy, CSP labeling would be achieved within 2 min of UV irradiation by developing a new photocrosslinking probe (SDB) followed by one-step enrichment. The enrichment selectivity of CSPs reached 70.5%, and we identified up to 1017 CSPs from living HEK-293T cells, attributed to the high photocrosslinking reactivity and inherent impermeability of SDB, as well as the high cell viability maintained after rapid cell surface labeling to decrease the interference of intracellular proteins. Finally, this strategy was successfully applied to sorafenib-resistance cells for quantitative surfaceome analysis.
All results demonstrated that our developed PCSPE method might provide a valuable toolkit for in-depth surfaceome profiling and comprehensive functional analysis.