Data-Independent Acquisition Mass Spectrometry Based Proteomic Analysis of Urinary Extracellular Vesicles Reveals Novel Candidate Biomarkers for Prostate Cancer

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles secreted by cells that carry diverse biomolecules, including proteins and nucleic acids, and have emerged as promising sources of disease biomarkers. In this study, an EVtrap-based enrichment strategy combined with data-independent acquisition mass spectrometry (DIA-MS) was established for comprehensive proteomic profiling of urinary EVs. Urine samples collected from six healthy controls and six patients with prostate cancer were subjected to EV isolation using EVtrap magnetic beads, followed by label-free DIA-MS analysis. A total of 3815 proteins and 63,122 peptides were identified in the healthy control group, whereas 3824 proteins and 64,174 peptides were identified in the prostate cancer group. Comparative analysis demonstrated that 91 identified proteins overlapped with the top 100 EV proteins reported in the Vesiclepedia database, confirming the high specificity and reliability of the EV enrichment workflow. Differential expression analysis revealed 78 significantly altered proteins, including 36 upregulated and 42 downregulated proteins in prostate cancer-derived urinary EVs. Further Kyoto encyclopedia of genes and genomes pathway enrichment analysis identified four proteins, MAP2K2, ATP5MC1, VDAC2, and VDAC3, that were closely associated with chemical carcinogenesis pathways. These findings suggest that urinary EV-associated proteins may serve as promising noninvasive biomarkers for prostate cancer and provide a valuable basis for early diagnosis and prognostic evaluation.