Isolation defines identity: Functional consequences of EV purification strategies

ABSTRACT: 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. Together, these findings demonstrate that even under standardized particle input, EV isolation workflows critically shape both EV proteomic profiles and functional activities. Our study provides a systematic, particle-normalized functional comparison of EV isolation methods, emphasizing the importance of workflow choice for biomarker discovery and mechanistic studies.

INSTRUMENT(S): timsTOF Ultra

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Elke Pogge von Strandmann  

PROVIDER: MSV000099567 | MassIVE | Thu Oct 23 02:30:00 BST 2025

SECONDARY ACCESSION(S): PXD069821

REPOSITORIES: MassIVE