Smolarz Mateusz, Pietrowska Monika, Matysiak Natalia, Mielańczyk Łukasz, Widłak Piotr
Maria Skłodowska-Curie Institute-Oncology Center, Gliwice Branch, 44-101 Gliwice, Poland.
Department of Histology and Cell Pathology, School of Medicine with Division of Dentistry in Zabrze, Medical University of Silesia, 41-800 Zabrze, Poland.
Proteomes. 2019 Apr 28;7(2):18. doi: 10.3390/proteomes7020018.
Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from a small amount of human serum (<1 mL) using the size-exclusion chromatography (SEC) standalone for the discovery of vesicle-specific proteins by the untargeted LC-MS/MS shotgun approach. We selected the SEC fraction containing vesicles with the size of about 100 nm and enriched with exosome markers CD63 and CD81 (but not CD9 and TSG101) and analyzed it in a parallel to the subsequent SEC fraction enriched in the lipoprotein vesicles. In general, there were 267 proteins identified by LC-MS/MS in exosome-containing fraction (after exclusion of immunoglobulins), yet 94 of them might be considered as serum proteins. Hence, 173 exosome-related proteins were analyzed, including 92 proteins absent in lipoprotein-enriched fraction. In this set of exosome-related proteins, there were 45 species associated with the GO cellular compartment term "extracellular exosome". Moreover, there were 31 proteins associated with different immune-related functions in this set, which putatively reflected the major role of exosomes released by immune cells present in the blood. We concluded that identified set of proteins included a bona fide exosomes components, yet the coverage of exosome proteome was low due to co-purified high abundant serum proteins. Nevertheless, the approach proposed in current work outperformed other comparable protocols regarding untargeted identification of exosome proteins and could be recommended for pilot exploratory studies when a small amount of a serum/plasma specimen is available.
由于从人血清或血浆中分离出的细胞外囊泡（EV）会被脂蛋白和其他丰富的血清成分污染，因此对其进行非靶向蛋白质组学分析仍然是一项技术挑战。在这里，我们旨在测试一种从少量人血清（<1 mL）中分离EV的简单方法，该方法使用尺寸排阻色谱法（SEC）单独进行，通过非靶向液相色谱-串联质谱（LC-MS/MS）鸟枪法发现囊泡特异性蛋白质。我们选择了含有大小约为100 nm且富含外泌体标志物CD63和CD81（但不包括CD9和TSG101）的囊泡的SEC馏分，并将其与随后富含脂蛋白囊泡的SEC馏分平行分析。一般来说，通过LC-MS/MS在含外泌体的馏分中鉴定出267种蛋白质（排除免疫球蛋白后），但其中94种可能被视为血清蛋白。因此，分析了173种与外泌体相关的蛋白质，包括在富含脂蛋白的馏分中不存在的92种蛋白质。在这组与外泌体相关的蛋白质中，有45种与GO细胞区室术语“细胞外囊泡”相关。此外，在这组蛋白质中有31种与不同的免疫相关功能相关，这可能反映了血液中存在的免疫细胞释放的外泌体的主要作用。我们得出结论，鉴定出的蛋白质组包含真正的外泌体成分，但由于共纯化的高丰度血清蛋白，外泌体蛋白质组的覆盖度较低。然而，当前工作中提出的方法在非靶向鉴定外泌体蛋白质方面优于其他可比方案，并且当有少量血清/血浆样本时，可推荐用于初步探索性研究。
