Division of Endocrinology, Department of Medicine, Mayo Clinic, 200 First Street SW, Joseph 5-194, Rochester, MN 55905, USA.
Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA.
Cell Rep Methods. 2021 Jul 26;1(3). doi: 10.1016/j.crmeth.2021.100055.
Extracellular vesicles (EVs) are released into blood from multiple organs and carry molecular cargo that facilitates inter-organ communication and an integrated response to physiological and pathological stimuli. Interrogation of the protein cargo of EVs is currently limited by the absence of optimal and reproducible approaches for purifying plasma EVs that are suitable for downstream proteomic analyses. We describe a size-exclusion chromatography (SEC)-based method to purify EVs from platelet-poor plasma (PPP) for proteomics profiling via high-resolution mass spectrometry (SEC-MS). The SEC-MS method identifies more proteins with higher precision than several conventional EV isolation approaches. We apply the SEC-MS method to identify the unique proteomic signatures of EVs released from platelets, adipocytes, muscle cells, and hepatocytes, with the goal of identifying tissue-specific EV markers. Furthermore, we apply the SEC-MS approach to evaluate the effects of a single bout of exercise on EV proteomic cargo in human plasma.
细胞外囊泡 (EVs) 从多个器官释放到血液中，并携带分子货物，促进器官间的通讯和对生理及病理刺激的综合反应。目前，对 EV 蛋白货物的检测受到缺乏合适和可重复的方法的限制，这些方法可用于纯化适合下游蛋白质组学分析的血浆 EV。我们描述了一种基于大小排阻色谱 (SEC) 的方法，用于从血小板贫乏血浆 (PPP) 中纯化 EV，通过高分辨率质谱 (SEC-MS) 进行蛋白质组学分析。SEC-MS 方法比几种传统的 EV 分离方法鉴定出更多具有更高精度的蛋白质。我们应用 SEC-MS 方法来鉴定来自血小板、脂肪细胞、肌肉细胞和肝细胞释放的 EV 的独特蛋白质组学特征，目的是鉴定组织特异性 EV 标志物。此外，我们应用 SEC-MS 方法评估单次运动对人血浆中 EV 蛋白质组货物的影响。
