A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum
A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum
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DOI：
10.1038/s41598-020-57497-7
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发表时间：
2020-01-23
影响因子：
4.6
通讯作者：
Mc Gee, M. M.
中科院分区：
文献类型：
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作者：
Brennan, K.;Martin, K.;Mc Gee, M. M.
细胞外囊泡（EV）是含有核酸和蛋白质货物的纳米尺寸囊泡，其从多种细胞类型释放，并且作为潜在的诊断生物标志物已经获得了显著的兴趣。人血清是容易获得的EV的丰富来源;然而，EV与血清蛋白和非EV脂质颗粒的分离代表了相当大的挑战。在这项研究中，我们比较了最常用的分离技术，无论是单独或组合，用于从200 μ l的人血清中分离EV，并将其与血清中存在的非EV蛋白和脂质颗粒分离。通过每种方法分离的颗粒的大小和产率通过纳米颗粒跟踪分析来确定，其中颗粒大小分布的变化用于确定脂蛋白和蛋白质聚集体对分离的EV群体的相对影响。通过计算EV颗粒计数与蛋白质浓度的比率来确定EV从可溶性蛋白质中的纯化。最后，与EV共分离的脂蛋白颗粒通过脂蛋白标志物APOB和APOE的蛋白质印迹分析来确定。总的来说，这项研究表明，EV分离程序的选择显着影响EV产量从人血清中，连同脂蛋白和蛋白质污染物的存在。
Extracellular vesicles (EVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers. Human serum is a rich source of readily accessible EVs; however, the separation of EVs from serum proteins and non-EV lipid particles represents a considerable challenge. In this study, we compared the most commonly used isolation techniques, either alone or in combination, for the isolation of EVs from 200 mu l of human serum and their separation from non-EV protein and lipid particles present in serum. The size and yield of particles isolated by each method was determined by nanoparticle tracking analysis, with the variation in particle size distribution being used to determine the relative impact of lipoproteins and protein aggregates on the isolated EV population. Purification of EVs from soluble protein was determined by calculating the ratio of EV particle count to protein concentration. Finally, lipoprotein particles co-isolated with EVs was determined by Western blot analysis of lipoprotein markers APOB and APOE. Overall, this study reveals that the choice of EV isolation procedure significantly impacts EV yield from human serum, together with the presence of lipoprotein and protein contaminants.