Size-Based Proteomic Signatures of Extracellular Vesicles Derived from Umbilical Cord Mesenchymal Stem Cells Fractionated by EXODUS

Umbilical cord mesenchymal stem cell-derived extracellular vesicles (UCMSC-EVs) hold strong promise for regenerative medicine, yet their intrinsic size heterogeneity remains a critical barrier to clinical translation, as it obscures molecular and functional specialization within bulk EV preparations. Here, we pioneer the application of the automated EXODUS platform to directly fractionate EVs from cell culture supernatants, resolving bulk UCMSC-EVs into three size-defined subpopulations. By integrating this platform with high-resolution mass spectrometry, we systematically characterize the molecular and functional landscapes of these UCMSC-EV size subpopulations. We demonstrate that EV size is tightly linked to distinct biogenetic origins, biomolecular corona composition, and functional programs: smaller EVs are enriched in exosome-associated proteins, ECM–glycan interfaces, and corona-associated molecules, and preferentially engage endocytosis- and phagosome-related pathways, whereas larger EVs exhibit ectosomal signatures. These findings identify EV size as a critical determinant of molecular architecture and biological function, providing insight into size-dependent EV heterogeneity and informing the rational design and optimization of UCMSC-EV-based therapeutic strategies.