Milk-derived exosomes have been reported, which are involved in many biological processes. The exosomes derived from mammary glands are not known yet, and their relationship with mammary gland lactation and the origin of milk-derived exosomes are largely unclear. The present study aimed to investigate the proteome of exosomes derived from bovine mammary epithelial cells (BMECs) and compare them with milk-derived exosomes in the database. BMEC-derived exosomes were successfully separated from the culture supernatant of BMECs by a combined ultracentrifugation approach, and the purity of exosomes was identified by western blot analysis. Liquid chromatography with tandem mass spectrometry identified 638 proteins in BMEC-derived exosomes. The MS data were deposited into the PUBLIC repository ProteomeXchange, dataset identifier(s): https://www.iprox.org/page/PSV023.html;?url=1590961453176tKpa. Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these proteins were associated with specific biological processes and molecular functions of metabolism. Cross comparison of these proteins with the protein database of milk exosomes showed that 77 common expressed proteins (CEPs) were in both BMEC- and milk-derived exosomes. The KEGG pathway analysis for these CEPs showed that they were mainly involved in signaling pathways associated with milk biosynthesis in BMECs. Among these CEPs, six proteins have been previously reported to be associated with the lactation function. The western blot analysis detected that expression of these six proteins in BMEC-derived exosomes was increased after the stimulation of methionine and β-estradiol on BMECs. In summary, the proteome of BMEC-derived exosomes reveals that they are associated with milk biosynthesis in BMECs and might be a source of milk-derived exosomes.

中文翻译：

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蛋白质组学分析揭示了与牛乳腺上皮细胞衍生外泌体泌乳相关的蛋白质和途径。

已经报道了牛奶来源的外​​泌体，其涉及许多生物学过程。尚不知道源自乳腺的外泌体，它们与乳腺泌乳和乳源性外泌体来源的关系尚不清楚。本研究旨在调查牛乳腺上皮细胞（BMEC）衍生的外泌体的蛋白质组，并将其与数据库中的牛奶来源的外​​泌体进行比较。通过联合超速离心方法成功地从BMEC的培养上清液中分离了BMEC衍生的外泌体，并通过Western blot分析鉴定了外泌体的纯度。液相色谱-串联质谱法鉴定了BMEC衍生的外泌体中的638种蛋白质。MS数据已存储到PUBLIC存储库ProteomeXchange中，数据集标识符为：https：//www.iprox。org / page / PSV023.html;？url = 1590961453176tKpa。基因本体论注释和《京都基因与基因组百科全书》（KEGG）途径分析表明，这些蛋白质与特定的生物过程和代谢的分子功能有关。这些蛋白质与牛奶外泌体蛋白质数据库的交叉比较显示，BMEC和牛奶来源的外​​泌体中共有77种共同表达的蛋白质（CEP）。这些CEP的KEGG通路分析表明，它们主要参与与BMEC中牛奶生物合成相关的信号通路。在这些CEP中，以前有6种蛋白质与哺乳功能有关。蛋白质印迹分析检测到，在BMEC上刺激蛋氨酸和β-雌二醇后，这六种蛋白在BMEC衍生的外泌体中的表达增加。