Circulating small extracellular vesicles (sEVs) and its associated proteins are of great interest in the early detection of many diseases. However, there is no gold standard for plasma sEVs isolation, especially for proteomic profiling which could be largely affected by contamination such as lipoproteins and plasma proteins. Previous studies suggested combinations of different sEVs isolation methods could improve the yield and purity of the isolated fractions. Nevertheless, there is no systematic evaluation of size-exclusion chromatography (SEC), ultracentrifugation (UC), and their combination in a proteomic perspective. Plasma samples were collected from healthy individuals, and sEVs were separated by one-step SEC, one-step UC, and combining SEC with UC, respectively. Here we exhibited that the purity of sEVs was improved by SEC in contrast to traditional UC. Furthermore, by conducting a SEC procedure followed by UC, we separated sEVs with the highest purity. In the proteomic analysis, 992 protein species were identified in the plasma sEVs isolated by our novel separation method, of which several proteins are sEVs-associated proteins but hitherto never been identified in the previous studies and database, much more than plasma sEVs isolated by UC (453) or SEC (682) alone. As compared to Vesiclepedia and Exocarta databases, plasma sEVs isolated by the new procedure kept 584 previously identified sEVs-associated proteins and 360 other proteins that have not been detected before. Detailed analysis suggested that more kinds of sEVs biomarkers, such as CD9, ALIX, and FLOT1, could be identified in plasma sEVs isolated by the novel isolation method as compared to one-step UC/SEC. Furthermore, the lower abundance ranks of common contaminants, such as lipoproteins and IgG chains, in the sEVs fractions obtained by our new method as compared to one-step UC/SEC also demonstrated the purity of sEVs had been improved. Combining SEC with UC could significantly improve the performance of mass spectrometry-based proteomic profiling in analyzing plasma-derived sEVs.

中文翻译：

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尺寸排阻色谱和超速离心相结合改进了血浆衍生的小细胞外囊泡的蛋白质组学分析。

循环小细胞外囊泡 (sEV) 及其相关蛋白在许多疾病的早期检测中具有重要意义。然而，血浆 sEV 的分离没有金标准，特别是蛋白质组学分析，这可能在很大程度上受到脂蛋白和血浆蛋白等污染的影响。先前的研究表明，不同 sEV 分离方法的组合可以提高分离组分的产量和纯度。然而，从蛋白质组学的角度来看，没有对尺寸排阻色谱 (SEC)、超速离心 (UC) 及其组合进行系统评估。血浆样本取自健康个体，sEVs 分别通过一步 SEC、一步 UC 和 SEC 与 UC 结合分离。在这里，我们展示了与传统 UC 相比，SEC 提高了 sEV 的纯度。此外，通过在 UC 之后进行 SEC 程序，我们分离了纯度最高的 sEV。在蛋白质组学分析中，通过我们的新分离方法在血浆 sEV 中鉴定出 992 种蛋白质，其中几种蛋白质是 sEV 相关蛋白，但迄今为止从未在之前的研究和数据库中发现，远远超过 UC 分离的血浆 sEV （453）或 SEC（682）单独。与 Vesiclepedia 和 Exocarta 数据库相比，通过新程序分离的血浆 sEV 保留了 584 种先前鉴定的 sEV 相关蛋白和 360 种以前未检测到的其他蛋白质。详细分析表明，更多种类的 sEV 生物标志物，如 CD9、ALIX 和 FLOT1，与一步 UC/SEC 相比，可以在通过新分离方法分离的血浆 sEV 中识别。此外，与一步 UC/SEC 相比，通过我们的新方法获得的 sEVs 组分中常见污染物（如脂蛋白和 IgG 链）的较低丰度等级也表明 sEVs 的纯度得到了提高。将 SEC 与 UC 相结合可以显着提高基于质谱的蛋白质组学分析在分析血浆衍生的 sEV 中的性能。