SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., 630090 Novosibirsk, Russia.
Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of Russian Academy of Sciences, 690022 Vladivostok, Russia.
Int J Mol Sci. 2019 May 16;20(10):2434. doi: 10.3390/ijms20102434.
Exosomes are nanovesicles (30-100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30-100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins' tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11-13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes.
外泌体是含有各种 RNA 和不同蛋白质的纳米囊泡（30-100nm）。外泌体在细胞内通讯、免疫功能等方面具有重要作用。来自不同来源（包括胎盘）的外泌体主要通过不同类型的离心和超速离心获得，据报道，它们含有几十到几千种不同的蛋白质。本研究首先使用几种标准离心法从四个胎盘（正常妊娠）中获得了粗制外泌体制剂，然后通过 Sepharose 4B 凝胶过滤进一步纯化。各个制剂显示出不同的凝胶过滤图谱，能够很好或很差地将外泌体峰与两种杂质蛋白峰及其复合物分离。根据电子显微镜，凝胶过滤前的外泌体包含不同大小的囊泡、由铁蛋白形成的环状结构以及聚集蛋白及其复合物的团簇。通过 220nm 过滤器和凝胶过滤后，外泌体显示出典型的外泌体形态和大小（30-100nm），并且不含可见的蛋白质杂质。通过 SDS-PAGE 和 2D 电泳后对蛋白质的胰蛋白酶水解物进行 MALDI 质谱和 MS/MS 分析，鉴定外泌体蛋白。我们得到了意想不到的结果。纯化的外泌体仅含有 11-13 种不同的蛋白质：CD9、CD81、CD-63、血红蛋白亚基、白细胞介素-1 受体、膜联蛋白 A1、膜联蛋白 A2、膜联蛋白 A5、细胞质肌动蛋白、碱性磷酸酶、转铁蛋白，可能还有人血清白蛋白和免疫球蛋白。我们假设，以前使用粗制制剂发现的外泌体蛋白数量可能被大大高估了。我们的数据对于研究纯外泌体的生物学功能可能很重要。
