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.