EV‐SPEC: Decoding the Spatial Proteomic Signature of the Extracellular Vesicle Corona

ABSTRACT The concept of protein corona formation around extracellular vesicles (EVs) has given birth to new insights into how cells may recognize EVs by proteins presented at the EV surface. Here we present spatially resolved proteomics using the biotin ligase TurboID to map proteins interacting at the EV surface, without a need for physical isolation of the EV‐corona complexes. TurboID promiscuously biotinylates nearby proteins within a few nm distance from the fused ‘bait’ protein. We genetically engineered EVs by modifying CD63 as the bait protein to which TurboID was fused facing outward to map EV corona proteins. Biotinylated proteins were then analysed by Western blotting and liquid chromatography‐mass spectrometry. Western blots revealed protein patterns that are distinct depending on the localization of the fused TurboID. The mass spectrometry analysis identified many of the serum proteins commonly known to form a corona around a synthetic solid nanoparticle, also supporting those previously reported through the physical isolation approaches. Rather striking is, however, the EV corona footprint of endogenous proteins that tells us about the EV biogenesis and what the intrinsic endogenous corona might look like. This approach, which we coined EV‐SPEC (Spatial Proteomics of Endogenous Corona), has thus the potential to revolutionize our understanding of EV biology by shifting the focus from the EVs themselves to the proteins that make up the corona, or how the cell ‘sees’ them, in analogy with the biomolecular corona extensively characterized for synthetic nanoparticles.