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Here, we compared four isolation strategies for EVs derived from malignant ascites and ES\u20102 ovarian cancer cell culture supernatants, assessing yield, particle size, protein cargo, and EV\u2010associated enzymatic activity. Proteomic analyses of particle\u2010normalized preparations were performed according to MISEV2023 guidelines, and vesicle\u2010associated protease activity was profiled using a FRET\u2010based assay with inhibitor panels. Principal component and overlap analyses identified a common EV proteome signature for ascites and ES\u20102 EVs, which was complemented by workflow\u2010dependent detection of additional proteins. Ultracentrifugation\/density gradient (UC\u2010DG) and tangential flow filtration\/size exclusion chromatography (TFF\u2010SEC) achieved the highest enrichment of canonical EV markers, whereas TFF\/ultrafiltration (TFF\u2010UF) was enriched in lipoproteins and secreted proteins. Functionally, UC\u2010DG and TFF\u2010SEC samples exhibited strong ADAM10\u2010associated activity, while TFF\u2010UF retained residual non\u2010metalloprotease activity. These results reveal to what extent EV purification methods impact both, EV composition and function. This methodological awareness is critical for advancing EV\u2010based biomarker discovery, diagnostics, and therapeutic platforms.<\/jats:p>","DOI":"10.1002\/adhm.202504684","type":"journal-article","created":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T07:06:19Z","timestamp":1768892779000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Isolation Defines Identity: Functional Consequences of Extracellular Vesicle Purification Strategies"],"prefix":"10.1002","volume":"15","author":[{"given":"Christian","family":"Preu\u00dfer","sequence":"first","affiliation":[{"name":"EV\u2010iTEC Core Facility Center for Tumor Biology and Immunology Marburg University  Marburg Germany"},{"name":"Institute for Tumor Immunology Center for Tumor Biology and Immunology Marburg University  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dolores 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Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kai","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery Marburg University  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J\u00f6rg W.","family":"Bartsch","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery Marburg University  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda","family":"G\u00f3mez\u2010Serrano","sequence":"additional","affiliation":[{"name":"Institute for Tumor Immunology Center for Tumor Biology and Immunology Marburg University  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Bachurski","sequence":"additional","affiliation":[{"name":"Department I of Internal Medicine Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf CECAD Center of Excellence on Cellular Stress Responses in Aging\u2010Associated Diseases Faculty of Medicine and University Hospital Cologne University of Cologne  Cologne Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silke","family":"Reinartz","sequence":"additional","affiliation":[{"name":"Translational Oncology Group Center for Tumor Biology and Immunology Marburg University  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4785-9165","authenticated-orcid":false,"given":"Elke","family":"von Pogge von Strandmann","sequence":"additional","affiliation":[{"name":"EV\u2010iTEC Core Facility Center for Tumor Biology and Immunology Marburg University  Marburg Germany"},{"name":"Institute for Tumor Immunology Center for Tumor Biology and Immunology Marburg University  Marburg 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