LAUSR

Abstract Extracellular vesicles (EVs) are blood‐borne messengers that coordinate signalling between different tissues and organs in the body. The specificity of such crosstalk is determined by preferential EV docking to target sites, as mediated through protein‐protein interactions. As such, the need to structurally characterize the EV surface precedes further understanding of docking selectivity and recipient‐cell uptake mechanisms. Here, we describe an intact extracellular vesicle crosslinking mass spectrometry (iEVXL) method that can be applied for structural characterization of protein complexes in EVs. By using a partially membrane‐permeable disuccinimidyl suberate crosslinker, proteins on the EV outer‐surface and inside EVs can be immobilized together with their interacting partners. This not only provides covalent stabilization of protein complexes before extraction from the membrane‐enclosed environment, but also generates a set of crosslinking distance restraints that can be used for structural modelling and comparative screening of changes in EV protein assemblies. Here we demonstrate iEVXL as a powerful approach to reveal high‐resolution information, about protein determinants that govern EV docking and signalling, and as a crucial aid in modelling docking interactions.