van Herwijnen Martijn J C, Zonneveld Marijke I, Goerdayal Soenita, Nolte-'t Hoen Esther N M, Garssen Johan, Stahl Bernd, Maarten Altelaar A F, Redegeld Frank A, Wauben Marca H M
From the ‡Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.
§Division of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, the Netherlands.
Mol Cell Proteomics. 2016 Nov;15(11):3412-3423. doi: 10.1074/mcp.M116.060426. Epub 2016 Sep 6.
Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome.
母乳含有几种具有独特功能的大分子成分，其中乳脂肪球和酪蛋白胶粒主要为新生儿提供营养，而乳清中含有能刺激新生儿免疫系统和胃肠道发育的分子。尽管母乳中已鉴定出细胞外囊泡（EV），但其生理功能和组成尚未得到详细研究。EV是细胞释放的亚微米大小的囊泡，通过选择性地整合脂质、核酸和蛋白质进行细胞间通讯。由于难以将EV与其他乳汁成分分离，因此缺乏对人乳来源EV蛋白质组的深入分析。在本研究中，使用最近建立的、优化的基于密度梯度的EV分离方案，对从7名个体捐赠者的母乳中纯化的EV进行了广泛的液相色谱-串联质谱（LC-MS/MS）蛋白质组分析。在乳汁来源的EV中总共鉴定出1963种蛋白质，包括与EV相关的蛋白质，如CD9、膜联蛋白A5和小窝蛋白-1，不同捐赠者之间有显著重叠。有趣的是，所鉴定的198种蛋白质在人类EV数据库Vesiclepedia中不存在，这表明乳汁来源的EV含有尚未在不同来源的EV中鉴定出的蛋白质。同样，将乳汁来源EV的蛋白质组与其他乳汁成分的蛋白质组进行了比较。为此，整合了38项已发表的乳汁蛋白质组研究的数据，以构建由2698种独特蛋白质组成的总乳汁蛋白质组。值得注意的是，在乳汁来源的EV中鉴定出的633种蛋白质迄今尚未在人乳中鉴定出来。有趣的是，这些新蛋白质包括参与细胞生长调节和控制炎症信号通路的蛋白质，这表明乳汁来源的EV可能支持新生儿胃肠道和免疫系统的发育。总体而言，本研究扩展了全乳汁蛋白质组，并表明乳汁来源的EV是具有独特功能蛋白质组的大分子成分。
