Isolation Defines Identity: Functional Consequences of Extracellular Vesicle Purification Strategies.

The biological activity of extracellular vesicles (EVs) is largely defined by their molecular cargo, yet the impact of isolation workflows on EV proteomes and function remains incompletely understood. Here, we compared four isolation strategies for EVs derived from malignant ascites and ES-2 ovarian cancer cell culture supernatants, assessing yield, particle size, protein cargo, and EV-associated enzymatic activity. Proteomic analyses of particle-normalized preparations were performed according to MISEV2023 guidelines, and vesicle-associated protease activity was profiled using a FRET-based assay with inhibitor panels. Principal component and overlap analyses identified a common EV proteome signature for ascites and ES-2 EVs, which was complemented by workflow-dependent detection of additional proteins. Ultracentrifugation/density gradient (UC-DG) and tangential flow filtration/size exclusion chromatography (TFF-SEC) achieved the highest enrichment of canonical EV markers, whereas TFF/ultrafiltration (TFF-UF) was enriched in lipoproteins and secreted proteins. Functionally, UC-DG and TFF-SEC samples exhibited strong ADAM10-associated activity, while TFF-UF retained residual non-metalloprotease activity. These results reveal to what extent EV purification methods impact both, EV composition and function. This methodological awareness is critical for advancing EV-based biomarker discovery, diagnostics, and therapeutic platforms.