Efficient capture and proteomic analysis of urinary extracellular vesicles by affinity purification

Liquid biopsy has become a promising alternative to traditional tissue biopsies for cancer diagnosis, offering advantages such as minimal invasiveness, accessibility, and ease of operation. Urinary extracellular vesicles (EVs) have emerged as an important source of cancer biomarkers. EVs are lipid bilayer vesicles secreted by cells, containing proteins, DNA, and RNA. The lipid bilayer protects EV proteins from degradation by enzymes present in body fluids. However, isolating EVs from biofluids with sufficient yield for proteomic analysis remains challenging. In this study, we employed EVlent, an affinity-based magnetic bead method, for selective enrichment of urinary EVs. This approach enables the high-specificity capture of EVs through surface protein recognition. We applied this method to urinary proteomic analysis in 15 healthy volunteers and 15 prostate cancer patients, identifying an average of 2039 proteins and 14,490 peptides in healthy controls, and 1982 proteins and 13,100 peptides in prostate cancer patients. Further analysis revealed 91 proteins commonly identified in the Vesiclepedia database. Compared to healthy controls, 88 proteins were upregulated and 90 were downregulated in prostate cancer patients. KEGG pathway analysis highlighted four proteins (PLAU, PDGFA, MMP3, and NRAS) enriched in the prostate cancer pathway, suggesting these proteins may serve as potential biomarkers for early diagnosis and prognosis of prostate cancer.