Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes
Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes
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DOI：
10.1073/pnas.1521230113
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发表时间：
2016-02-23
影响因子：
11.1
通讯作者：
Thery, Clotilde
中科院分区：
文献类型：
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作者：
Kowal, Joanna;Arras, Guillaume;Thery, Clotilde
细胞外囊泡（EVs）由于其在生理和病理上的众多功能而成为研究的热点。细胞释放不同大小和细胞内起源的异质囊泡，包括在内体区室内部形成的小EV（即，外泌体）和从质膜出芽的各种大小的EV。用于分析和分离不同EV人群的特定标志物缺失，对理解EV功能施加了重要限制。在这里，来自人树突细胞的EV首先通过其沉降速度分离，然后通过其向上漂浮到碘克沙醇梯度中时的行为或通过免疫分离分离。广泛的定量蛋白质组学分析，允许比较分离的人群显示，几个经典使用的外泌体标记物，如主要组织相容性复合体，flotillin，和热休克70 kDa蛋白，类似地存在于所有EV。我们鉴定了在小EV中特异性富集的蛋白质，并定义了一组在不同EV群体中显示不同相对丰度的五种蛋白质类别。我们证明了小EV内存在外泌体和非外泌体亚群，并提出了使用CD63，CD81或CD9通过免疫分离进行差异分离。因此，我们的工作提供了指导方针，以确定亚型的EV为未来的功能研究。
Extracellular vesicles (EVs) have become the focus of rising interest because of their numerous functions in physiology and pathology. Cells release heterogeneous vesicles of different sizes and intracellular origins, including small EVs formed inside endosomal compartments (i.e., exosomes) and EVs of various sizes budding from the plasma membrane. Specific markers for the analysis and isolation of different EV populations are missing, imposing important limitations to understanding EV functions. Here, EVs from human dendritic cells were first separated by their sedimentation speed, and then either by their behavior upon upward floatation into iodixanol gradients or by immuno-isolation. Extensive quantitative proteomic analysis allowing comparison of the isolated populations showed that several classically used exosome markers, like major histocompatibility complex, flotillin, and heat-shock 70-kDa proteins, are similarly present in all EVs. We identified proteins specifically enriched in small EVs, and define a set of five protein categories displaying different relative abundance in distinct EV populations. We demonstrate the presence of exosomal and nonexosomal subpopulations within small EVs, and propose their differential separation by immuno-isolation using either CD63, CD81, or CD9. Our work thus provides guidelines to define subtypes of EVs for future functional studies.