将癌细胞信号转化成临床领域中的研究能力和知识的翻译一直缓慢而且效果不显著。近期，细胞外囊泡（EVs）作为一种有前途的来源出现，用于开发疾病磷酸化蛋白标记物以监测疾病的状态。本研究的重点是开发一种可靠的数据无关采集（DIA），利用质谱对尿液EV磷蛋白组学进行分析以区分肾细胞癌（RCC）的等级。我们研究了气相分离（GPF）文库、直接无库DIA、禁止区域和几个不同的窗口策略。在开发EV磷蛋白组学的DIA质谱方法之后，我们将该策略应用于鉴定和量化代表低级别透明细胞RCC、高级别透明细胞RCC、慢性肾脏病（CKD）和健康控制（HC）人群的57个个体的尿液EV磷蛋白质组。尿液EVs通过功能性磁珠高效分离，EV磷酸肽随后通过PolyMAC富集。我们量化了2584个独特的磷酸位点，并发现多个显著的与癌症相关的通路，如ErbB信号通路、肾脏细胞癌和肌动蛋白细胞骨架的调节等，仅在高级别透明细胞RCC中上调。这些结果表明，利用我们优化的EV分离、磷肽富集和DIA方法的EV磷蛋白质组分析为未来的临床应用提供了一个强大的工具。版权所有©2023年作者。由Elsevier公司出版。保留所有权利。

Translating the research capability and knowledge in cancer signaling into clinical settings has been slow and ineffective. Recently, extracellular vesicles (EVs) have emerged as a promising source for developing disease phosphoprotein markers to monitor disease status. This study focuses on the development of a robust data-independent acquisition (DIA) using mass spectrometry to profile urinary EV phosphoproteomics for renal cell cancer (RCC) grades differentiation. We examined gas-phase fractionated (GPF) library, direct DIA (library-free), forbidden zones, and several different windowing schemes. After the development of a DIA mass spectrometry method for EV phosphoproteomics, we applied the strategy to identify and quantify urinary EV phosphoproteomes from 57 individuals representing low-grade clear cell RCC, high-grade clear cell RCC, chronic kidney disease (CKD), and healthy control (HC) individuals. Urinary EVs were efficiently isolated by functional magnetic beads, and EV phosphopeptides were subsequently enriched by PolyMAC. We quantified 2,584 unique phosphosites and observed that multiple prominent cancer-related pathways, such as ErbB signaling, renal cell carcinoma, and regulation of actin cytoskeleton, were only upregulated in high-grade clear cell RCC. These results show that EV phosphoproteome analysis utilizing our optimized procedure of EV isolation, phosphopeptide enrichment, and DIA method provides a powerful tool for future clinical applications.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.