Feasibility of urinary extracellular vesicle proteome profiling using a robust and simple, clinically applicable isolation method

Extracellular vesicles (EVs) secreted by prostate cancer (PCa) cells contain specific biomarkers and can be isolated from urine. Collection of urine is not invasive, and therefore urinary EVs represent a liquid biopsy for diagnostic and prognostic testing for PCa. In this study, we optimised urinary EV isolation using a method based on heat shock proteins and compared it to gold-standard ultracentrifugation. The urinary EV isolation protocol using the Vn96-peptide is easier, time convenient (approximate to 1.5 h) and no special equipment is needed, in contrast to ultracentrifugation protocol (> 3.5 h), making this protocol clinically feasible. We compared the isolated vesicles of both ultracentrifugation and Vn96-peptide by proteome profiling using mass spectrometry-based proteomics (n = 4 per method). We reached a depth of > 3000 proteins, with 2400 proteins that were commonly detected in urinary EVs from different donors. We show a large overlap (> 85%) between proteins identified in EVs isolated by ultracentrifugation and Vn96-peptide. Addition of the detergent NP40 to Vn96-peptide EV isolations reduced levels of background proteins and highly increased the levels of the EV-markers TSG101 and PDCD6IP, indicative of an increased EV yield. Thus, the Vn96-peptide-based EV isolation procedure is clinically feasibly and allows largescale protein profiling of urinary EV biomarkers.