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In this study, small seminal EVs were isolated from healthy donors by size\u2010exclusion chromatography (SEC) and three ultracentrifugation (UC) protocols, with or without filtration. EV samples prepared by UC had a higher abundance of typical EV\u2010related molecules such as CD9, CD81, and Alix, compared to SEC preparations. However, ribosomal and ER\u2010related proteins were more enriched in SEC samples. Furthermore, the level of several nonvesicular proteins and\/or EV\u2010corona proteins was higher in SEC samples. The addition of a filtration step in the UC protocols also reduced the abundance of these proteins. Finally, we found that the isolation protocol affected the presence of prostate cancer\u2010specific proteins in the EV samples, with membrane proteins such as FOLH1, TMPRSS2, and PSCA enriched in samples prepared by UC. In conclusion, our results suggest that SEC and UC enrich for different EV populations, which should be considered when analyzing cancer biomarkers. Moreover, UC samples contained fewer nonvesicular proteins and\/or EV\u2010corona proteins, which may enable a more precise detection of EV\u2010based cancer biomarkers.<\/jats:p>","DOI":"10.1002\/1878-0261.70345","type":"journal-article","created":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T10:21:49Z","timestamp":1792664509000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimization of seminal extracellular vesicle isolation protocols for cancer biomarker analysis using proteomic profiling"],"prefix":"10.1002","author":[{"given":"Elen","family":"Telumyan","sequence":"first","affiliation":[{"name":"Department of Circulation and Medical Imaging NTNU  Trondheim Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Beatriz","family":"Mart\u00edn\u2010Gracia","sequence":"additional","affiliation":[{"name":"Department of Molecular Cell Biology, Institute for Cancer Research Oslo 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