A High-Resolution Proteomic Atlas of Extracellular Vesicles and Non-Vesicular Particles

Extracellular vesicles (EVs) are key mediators of intercellular communication and promising biomarkers. However, their molecular characterization remains challenging due to the heterogeneity of EV subtypes and co-isolated nanoparticles. Here, leveraging protein correlation profiling along density gradients, we systematically analyze over 9,000 proteins in human cancer cell lines and biofluids, providing a rigorous reassessment of virtually all protein constituents associated with small EVs (sEVs) and non-vesicular entities. We reveal that sEVs primarily incorporate plasma membrane proteins via selective cargo-loading mechanisms, with minimal inclusion of intraluminal soluble proteins. In contrast, the abundant cytosolic proteins frequently detected in sEVs preparations are not encapsulated within sEVs but bound externally, resulting from extracellular contamination by cell debris and intracellular interactions with autophagic material. Our work provides a reference resource for understanding the biogenesis, molecular determinants of cargo selection and functional roles of sEVs.