Salivary extracellular vesicles (EVs), as novel functional carriers and potential biomarkers, are usually obtained by ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation methods. However, salivary EVs obtained by these two methods have not been systematically compared. Here, we perform an in-depth analysis on EVs isolated by these two methods using proteomics. Both methods obtain EVs ranging from 40 to 210 nm, with the PEG method resulting in a wider size distribution. PEG-separated products were irregularly shaped and aggregated, while UC-separated ones were monodispersed and teacup-shaped. Additionally, the expression of EV-specific markers was higher in UC-separated EVs. Using tandem mass spectrometry proteomics, we identified and quantified 1217 kinds of saliva exosomal proteins and 361 kinds of differential proteins, showing that UC can isolate more EV-related proteins. These results offer some guidance for EV separating and provide potential direction for the use of EVs in non-invasive diagnosis.

唾液细胞外囊泡 (EV) 作为新型功能载体和潜在的生物标志物，通常通过超速离心 (UC) 和基于聚乙二醇 (PEG) 的沉淀方法获得。然而，通过这两种方法获得的唾液 EV 尚未系统地进行比较。在这里，我们使用蛋白质组学对这两种方法分离的 EV 进行了深入分析。这两种方法都能获得 40 到 210 nm 的 EV，而 PEG 方法导致更宽的尺寸分布。PEG分离的产物呈不规则形状和聚集状，而UC分离的产物呈单分散状和茶杯状。此外，在 UC 分离的 EV 中，EV 特异性标志物的表达更高。使用串联质谱蛋白质组学，我们鉴定并定量了1217种唾液外泌体蛋白和361种差异蛋白，表明 UC 可以分离出更多的 EV 相关蛋白。这些结果为EV分离提供了一些指导，并为EV在非侵入性诊断中的使用提供了潜在的方向。