Exosomes from human placenta purified by affinity chromatography on sepharose bearing immobilized antibodies against CD81 tetraspanin contain many peptides and small proteins
Exosomes from human placenta purified by affinity chromatography on sepharose bearing immobilized antibodies against CD81 tetraspanin contain many peptides and small proteins
复制标题
DOI：
10.1002/iub.1928
复制
发表时间：
2018-11-01
期刊：
影响因子：
4.6
通讯作者：
Nevinsky, Georgy A.
中科院分区：
文献类型：
--
作者：
Burkova, Evgeniya E.;Dmitrenok, Pavel S.;Nevinsky, Georgy A.
外泌体是含有多种rna和不同蛋白质的纳米囊泡（40-100 nm）。外泌体参与细胞内通讯和免疫系统功能。来自不同来源的外泌体通常使用标准的离心和超离心方法分离。据报道，通过这些方法分离的外泌体含有几十到几千种不同的蛋白质。本研究首先使用标准离心程序获得5个正常妊娠胎盘的粗泡制备物。电镜研究表明，这些制备中含有不同大小的囊泡（30-225 nm），平均电子密度为圆形的颗粒（20-40 nm的非囊泡）(A)，相关蛋白的结构簇和无形状聚集(B)，以及铁蛋白形成的10-14 nm的环状结构(C)。通过Sepharose 4B凝胶过滤对囊泡进行额外纯化后，去除了主要的蛋白质结构；然而，这些制剂仍然含有少量的组分A-C的外加剂。进一步用亲和层析法对该制剂进行纯化，得到了高度纯化的外泌体（40-100 nm）。根据电镜数据，这些外泌体含有包埋在膜上的tetraspanin，并用10-12 nm金纳米颗粒结合的CD81抗体对其进行染色。SDS-PAGE和MALDI质谱和MS/MS质谱分析显示，这些外泌体中含有的11种主要蛋白（bbb10 kDa）：血红蛋白亚基、CD81、白细胞介素-1受体、膜联蛋白A5、细胞质肌动蛋白、α -肌动蛋白-4、碱性磷酸酶、人血清白蛋白、血清转铁蛋白和乳铁蛋白。通过对高纯度外泌体的MALDI质量分析，我们首次发现除了大蛋白（bbb10 kDa）外，与CD81有亲和力的外泌体含有超过27种不同的肽和2-10 kDa的小蛋白。这一发现有助于揭示纯外泌体的生物学功能。(c)地球物理学报，2018,30 (11):1144-1155,2018
Exosomes are nanovesicles (40-100 nm) containing various RNAs and different proteins. Exosomes are involved in intracellular communication and immune system function. Exosomes from different sources are usually isolated using standard methodscentrifugation and ultracentrifugations. Exosomes isolated by these procedures were reported to contain from a few dozen to thousands of different proteins. Here crude vesicle preparations from five placentas (normal pregnancy) were first obtained using standard centrifugation procedures. According to electron-microscopic studies, these preparations contained vesicles of different size (30-225 nm), particles of round shape of average electron density (nonvesicles 20-40 nm) (A), structured clusters of associated proteins and shapeless aggregations (B), as well as ring-shaped 10-14 nm structures formed by ferritin (C). After additional purification of the vesicle preparations by gel filtration on Sepharose 4B, the main part of protein structures was removed; however, the preparations still contained small admixtures of components A-C. Further purification of the preparations by affinity chromatography on Sepharose bearing immobilized antibodies against exosome surface protein CD81 led to isolation of highly purified exosomes (40-100 nm). These exosomes according to electron microscopy data contained tetraspanin embedded in the membrane, which was stained with antibodies against CD81 conjugated with 10-12 nm gold nanoparticles. SDS-PAGE and MALDI MS and MS/MS mass spectrometry of tryptic hydrolysates of proteins contained in these exosomes revealed eleven major proteins (>10 kDa): hemoglobin subunits, CD81, interleukin-1 receptor, annexin A5, cytoplasmic actin, alpha-actin-4, alkaline phosphatase, human serum albumin, serotransferrin, and lactotrasferrin. Using MALDI mass analysis of the highly purified exosomes, we for the first time found that in addition to the large proteins (>10 kDa), exosomes having affinity to CD81 contain more than 27 different peptides and small proteins of 2-10 kDa. This finding can be useful for revealing biological functions of pure exosomes. (c) 2018 IUBMB Life, 70(11):1144-1155, 2018