细胞外囊泡（EV）是由活细胞释放的脂质膜囊泡，携带各种生物分子，包括核酸、脂质和蛋白质。近年来，血浆来源的EV中的蛋白质已成为新型生物标志物，在人类疾病的诊断和预后中起着重要作用。然而，目前从血浆中分离EV的方法常常导致生物液中共同分离的杂质。因此，在执行任何研究方案之前，必须优化从血浆中提取EV进行蛋白质组学分析的过程。本研究比较了两种EV分离策略，尺寸排除色谱（SEC）和SEC与离子交换吸附（SEC + IEA）相结合，从纯度和EV蛋白质数量方面进行了比较。我们的结果表明，与单步SEC相比，SEC与IEA相结合能够通过降低脂蛋白的含量来产生纯度更高的血浆来源EV。此外，通过MS分析，我们证明了这种组合方法在许多血浆样本中保持了EV的稳定性和提高了纯度。此外，通过结合SEC和IEA，检测到更多与癌症相关的蛋白质。©2023 Wiley-VCH GmbH。

Extracellular vesicles (EVs) are lipid membrane vesicles released by live cells that carry a variety of biomolecules, including nucleic acids, lipids, and proteins. Recently, proteins in plasma-derived EVs have emerged as novel biomarkers with essential functions in the diagnosis and prognosis of human diseases. However, the current methods of isolating EVs from plasma often lead to coisolated impurities in biological fluids. Therefore, before performing any research protocol, the process of extracting EVs from plasma for proteomic analysis must be optimized. In this study, two EV isolation strategies, size exclusion chromatography (SEC) and SEC combined with ion exchange adsorption (SEC + IEA), were compared in terms of the purity and quantity of protein in EVs. Our results demonstrated that, compared to single-step SEC, SEC combined with IEA could produce plasma-derived EVs with a higher purity by decreasing the abundance of lipoprotein. Additionally, with MS analysis, we demonstrated that the combination approach maintained the stability and improved the purity of EVs in many plasma samples. Furthermore, by combining SEC with IEA, more cancer-associated proteins were detected in the plasma of various cancer samples.© 2023 Wiley-VCH GmbH.