Spatiotemporally Resolved Tracking of Extracellular Vesicle Cargo via Photocatalytic Extracellular Vesicles Protein Tracking (PhoEVT).

Extracellular vesicle (EV)-mediated protein transport has been recognized as a crucial mechanism for intercellular communication and interaction. Proximity labeling has emerged as a promising approach to study the dynamic protein interactions in cellular activity, but tracking extracellular protein transport pathways remains to be further investigated. Here, we employ Ru(bpy)3 2+ (RU) to construct a spatiotemporally resolved Photocatalytic Extracellular Vesicles protein Tracking (PhoEVT) system. Upon 450 nm irradiation, RU generates singlet oxygen to trigger the biotinylation of EV-associated proteins with high efficiency. The photocatalyst was used as a nongenetic alternative to enzymatic approaches for singlet oxygen generation upon 450 nm photoirradiation during EV purification, which allowed the subsequent labeling of nearby accessible proteins with the biotin-aniline probe. Using spintip streptavidin-mediated affinity purification followed by mass spectrometry, we tracked EV-associated protein signals and detected EV-derived proteins in recipient cells. Based on the PhoEVT system, we demonstrated the uptake dynamics of EV-associated proteins in recipient cells and performed unbiased proteomic profiling of detected EV-derived proteins. Furthermore, we monitored the presence and temporal changes of EV-associated proteins during the interaction of tumor cell-derived EVs with immune cells, providing an effective tool to better investigate the dynamics of EV-associated proteins.