Integrated multi‐biofluid extracellular vesicle isolation with dual‐platform mass spectrometry metabolomics for prostate cancer detection

Abstract Prostate cancer poses a significant clinical challenge due to limited screening methods and incomplete understanding of its metabolic rewiring. Extracellular vesicles (EVs) carry bioactive metabolites that mirror tumor biology, enabling mechanistic study and biomarker discovery. We evaluated five EV isolation methods and determined that ultracentrifugation and size‐exclusion chromatography (SEC) yielded relatively higher EV recovery and purity. In expanded cohorts (plasma n = 36; urine n = 44), these methods were paired with liquid chromatography–mass spectrometry and laser desorption/ionization–mass spectrometry to profile EV metabolic fingerprints. Integration with single‐cell RNA sequencing and pathway enrichment highlighted palmitic and stearic acids as key metabolites linked to clinical parameters and fatty acid biosynthesis. Diagnostic models built on these workflows achieved ​area under the curves above 0.85, with the plasma + SEC + LDI‐MS combination offering the best trade‐off between accuracy, speed, and cost. This scalable EV‐metabolomics pipeline promises prostate cancer detection and mechanistic insight for broader liquid biopsy applications.