细胞外囊泡（EV）蛋白质组学成为发现疾病诊断、监测和治疗潜在生物标志物的有效工具。然而，基于质谱的EV蛋白质组分析的当前工作流程由于低效的EV分离方法和繁琐的样品制备过程，在临床环境中并不完全兼容。为了简化和提高EV蛋白质组分析的效率，我们在这里引入了一种一锅式分析流程，将稳健的EV分离方法EVtotal recovery and purification (EVtrap)与原位蛋白样品制备相结合，用于检测尿液EV蛋白质组。通过结合溶剂驱动的蛋白质捕获和快速的颗粒上消化，一锅式分析流程使整个EV蛋白质组分析能够在一天内完成。与现有工作流程相比，一锅式分析流程能够获得更好的肽收率，并从1 mL尿液中鉴定出同等数量的独特EV蛋白质。最后，我们将一锅式分析流程应用于膀胱癌患者尿液EV的蛋白组学分析。使用15分钟梯度的无库存数据无目标采集方法，在53个尿液样本中鉴定出了2774种独特蛋白质。总之，我们的新型一锅式分析流程展示了在生物医学应用中进行日常和稳健的EV蛋白质组学的潜力。

Extracellular vesicle (EV) proteomics emerges as an effective tool for discovering potential biomarkers for disease diagnosis, monitoring, and therapeutics. However, the current workflow of mass spectrometry-based EV proteome analysis is not fully compatible in a clinical setting due to inefficient EV isolation methods and a tedious sample preparation process. To streamline and improve the efficiency of EV proteome analysis, here we introduce a one-pot analytical pipeline integrating a robust EV isolation approach, EV total recovery and purification (EVtrap), with in situ protein sample preparation, to detect urinary EV proteome. By incorporating solvent-driven protein capture and fast on-bead digestion, the one-pot pipeline enabled the whole EV proteome analysis to be completed within one day. In comparison with the existing workflow, the one-pot pipeline was able to obtain better peptide yield and identify the equivalent number of unique EV proteins from 1 mL of urine. Finally, we applied the one-pot pipeline to profile proteomes in urinary EVs of bladder cancer patients. A total of 2774 unique proteins were identified in 53 urine samples using a 15 min gradient library-free data-independent acquisition method. Taken altogether, our novel one-pot analytical pipeline demonstrated its potential for routine and robust EV proteomics in biomedical applications.