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 (≈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 large-scale protein profiling of urinary EV biomarkers.

前列腺癌（PCa）细胞分泌的细胞外囊泡（EVs）包含特定的生物标志物，可以从尿液中分离出来。尿液的收集不是侵入性的，因此尿液电动车代表液体活检，用于PCa的诊断和预后测试。在这项研究中，我们使用基于热激蛋白的方法优化了尿液电动隔离，并将其与金标准超速离心法进行了比较。与超速离心协议（> 3.5 h）相比，使用Vn96肽的尿液EV隔离协议更容易，更方便（约1.5小时）并且不需要专用设备，该协议在临床上是可行的。我们比较了使用基于质谱的蛋白质组学通过蛋白质组分析对超速离心和Vn96肽分离的囊泡（n =每个方法4个）。我们达到了> 3000种蛋白质的深度，其中2400种蛋白质通常是在来自不同供体的尿液电动汽车中检测到的。我们显示通过超速离心分离的EV和Vn96肽中鉴定出的蛋白质之间存在较大的重叠（> 85％）。将去污剂NP40添加到Vn96肽EV分离物中可降低背景蛋白的水平，并大大提高EV标记TSG101和PDCD6IP的水平，这表明EV产量增加。因此，基于Vn96肽的EV分离程序在临床上是可行的，并且可以对尿EV生物标志物进行大规模的蛋白质分析。