分子伴侣是广泛存在的细胞内蛋白质，通过协助客体蛋白的折叠起到维持蛋白质组功能的重要作用。分子伴侣，如热休克蛋白（HSPs），在细胞内和细胞外都可找到。细胞外囊泡（EVs），如外泌体，包含HSPs，并通过水平转运这些功能性伴侣至各种受体细胞。此外，质谱技术使得外泌体和EV蛋白质的全面分析成为可能，这对于基础生物医学研究以及临床生物标志物的搜索非常有用。我们对转移性舌癌和前列腺癌的EVs，包括外泌体在内，进行了深度蛋白质组学分析，检测到了700余种蛋白质类型，其中包括细胞质、内质网、线粒体、小型和大型HSPs。在这里，我们提供了分离外泌体/EV和深度蛋白质组学分析以检测EV伴侣的相关实验步骤。© 2023作者(们)，在Springer Science+Business Media, LLC，属于Springer Nature独家许可的情况下。

Molecular chaperones are widely distributed intracellular proteins that play essential roles in maintaining proteome function by assisting in the folding of client proteins. Molecular chaperones, such as heat shock proteins (HSPs), are found intracellularly and extracellularly. Extracellular vesicles (EVs), such as exosomes, contain HSPs and horizontally transfer the functional chaperones into various recipient cells. Besides, mass spectrometry has enabled a comprehensive analysis of exosomal and EV proteins, which is useful in basic biomedical research to clinical biomarker search. We have performed deep proteome analysis of EVs, including exosomes, from metastatic tongue and prostate cancers and detected >700 protein types, including cytoplasmic, ER, mitochondrial, small, and large HSPs. Here, we provide protocols for isolating exosomes/EVs and deep proteome analysis to detect the EV chaperone.© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.