Sedykh Sergey E, Purvinish Lada V, Monogarov Artem S, Burkova Evgeniya E, Grigor'eva Alina E, Bulgakov Dmitrii V, Dmitrenok Pavel S, Vlassov Valentin V, Ryabchikova Elena I, Nevinsky Georgy A
SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., Novosibirsk 630090, Russia.
Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russia.
Biochim Open. 2017 Mar 1;4:61-72. doi: 10.1016/j.biopen.2017.02.004. eCollection 2017 Jun.
Exosomes are 40-100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel filtration. Individual preparations demonstrated different profiles of gel filtration showing well or bad separation of exosome peaks and one or two peaks of co-isolating proteins and their complexes. According to the electron microscopy, well purified exosomes displayed a typical exosome-like size (30-100 nm) and morphology. It was shown that exosomes may have several different biological functions, but detection of their biological functions may vary significantly depending on the presence of exosome contaminating proteins and proteins directly into exosomes. Exosome proteins were identified before and after gel filtration by MALDI MS and MS/MS spectrometry of protein tryptic hydrolyzates derived by SDS PAGE and 2D electrophoresis. The results of protein identification were unexpected: one or two peaks co-isolating proteins after gel-filtration mainly contained kappa-, beta-, alpha-S1-caseins and its precursors, but these proteins were not found in well-purified exosomes. Well-purified exosomes contained from five to eight different major proteins: CD81, CD63 receptors, beta-lactoglobulin and lactadherin were common to all preparations, while actin, butyrophilin, lactoferrin, and xanthine dehydrogenase were found only in some of them. The article describes the morphology and the protein content of major horse milk exosomes for the first time. Our results on the decrease of major protein number identified in exosomal preparations after gel filtration may be important to the studies of biological functions of pure exosomes.
外泌体是40 - 100纳米的纳米囊泡，含有RNA和不同的蛋白质。含有蛋白质、脂质、信使核糖核酸和微小核糖核酸的外泌体在细胞内通讯和免疫功能中很重要。不同来源的外泌体通常通过离心和超速离心相结合的方法获得，根据已发表的数据，其可含有几十到数千种不同的蛋白质。首次通过多次标准离心从18匹马的乳汁中获得了粗制外泌体制剂。外泌体制剂还通过快速蛋白质液相色谱（FPLC）凝胶过滤进行了进一步纯化。各个制剂显示出不同的凝胶过滤图谱，外泌体峰的分离效果有好有坏，还有一两个共分离蛋白质及其复合物的峰。根据电子显微镜观察，纯化良好的外泌体呈现出典型的外泌体样大小（30 - 100纳米）和形态。结果表明，外泌体可能具有几种不同的生物学功能，但根据外泌体污染蛋白和直接进入外泌体的蛋白的存在情况，其生物学功能的检测可能会有显著差异。通过基质辅助激光解吸电离质谱（MALDI MS）和串联质谱（MS/MS）对十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS PAGE）和二维电泳衍生的蛋白质胰蛋白酶水解产物进行光谱分析，对外泌体蛋白在凝胶过滤前后进行了鉴定。蛋白质鉴定结果出乎意料：凝胶过滤后共分离蛋白质的一两个峰主要含有κ -、β -、α - S1 -酪蛋白及其前体，但在纯化良好的外泌体中未发现这些蛋白质。纯化良好的外泌体含有5至8种不同的主要蛋白质：CD81、CD63受体、β -乳球蛋白和乳黏附素在所有制剂中都很常见，而肌动蛋白、嗜乳脂蛋白、乳铁蛋白和黄嘌呤脱氢酶仅在其中一些制剂中发现。本文首次描述了马奶主要外泌体的形态和蛋白质含量。我们关于凝胶过滤后外泌体制剂中鉴定出的主要蛋白质数量减少的结果，可能对纯外泌体生物学功能的研究具有重要意义。
