[Efficient capture and proteomics analysis of urinary extracellular vesicles by affinity purification].

Liquid biopsy is a promising alternative to traditional tissue biopsies for diagnosing cancer because it offers advantages such as minimal invasiveness, accessibility, and ease of operation. Extracellular vesicles (EVs) are lipid bilayer vesicles that contain proteins, DNA, and RNA and are secreted by cells. Indeed, urinary EVs are important sources of cancer biomarkers. The lipid bilayer protects EV proteins from degradation by enzymes present in bodily fluids. Prostate cancer (PCa) is among the most prevalent malignancies in developed countries and is the second-leading cause of cancer-related mortality in men. Current screening methods commonly used to initially evaluate patients with suspected PCa include serum prostate-specific antigen (PSA) testing and digital rectal examination (DRE), with magnetic resonance imaging (MRI) and transrectal ultrasound often recommended for further assessment. However, both PSA testing and DRE have limited specificities, which results in a substantial number of unnecessary prostate biopsies. Consequently, additional reliable biomarkers need to be urgently discovered for rapidly diagnosing PCa more accurately. Prostate-derived secretions, including those associated with malignancies, are detectable in urine owing to the anatomical proximity of the prostate to the urethra; hence urine is a promising liquid-biopsy medium for discovering PCa biomarkers, which is a topic that has been the focus of extensive research efforts in recent years. However, isolating EVs from biofluids in sufficient yields for proteomics analysis remains challenging. In this study, functional magnetic beads EVlent (extracellular vesicles isoLated efficiently, naturally, and totally) with high-affinity capabilities were developed for selectively enriching EVs from biological fluids.The surfaces of the beads were modified with three antibodies that target CD9, CD63, and CD81, which enables the specific recognition of EV surface proteins. The isolation performance of EVlent was validated by comprehensively characterizing urinary EVs using Western blotting (WB), nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). WB revealed prominent bands for EV markers (CD9, TSG101, and HSP70) in EVlent-enriched samples, whereas weaker bands were observed following ultracentrifugation (UC). NTA revealed that the EVs isolated by EVlent are predominantly in the 50-400 nm size range, with a content of 4.1×10