A novel affinity-based method for the isolation of highly purified extracellular vesicles.
A novel affinity-based method for the isolation of highly purified extracellular vesicles.
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DOI：
10.1038/srep33935
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发表时间：
2016-09-23
影响因子：
4.6
通讯作者：
Hanayama R
中科院分区：
文献类型：
--
作者：
Nakai W;Yoshida T;Diez D;Miyatake Y;Nishibu T;Imawaka N;Naruse K;Sadamura Y;Hanayama R
细胞外囊泡（EV）如外来体和微囊泡充当细胞间网络的信使，允许细胞组分在细胞之间交换。EV携带来自其生产细胞的脂质，蛋白质和RNA，并具有作为细胞类型甚至细胞状态特异性生物标志物的潜力。然而，用于分离EV的常规方法（例如超离心或聚合物沉淀）在纯度和可行性方面具有缺点。在这里，我们已经开发了一种新的EV纯化方法，通过使用Tim4蛋白，它特异性结合的磷脂酰丝氨酸显示在EV的表面上。由于结合是Ca2+依赖性的，因此通过添加Ca2+螯合剂可以容易地从Tim4释放完整的EV。我们已经将Tim4纯化应用于细胞条件培养基和生物流体，能够产生比使用常规方法获得的EV更高纯度的EV。在Tim4纯化的EV制剂中发现的较低污染允许通过质谱法检测更多EV特异性蛋白，从而能够更好地表征和定量不同EV群体的蛋白质组。Tim4蛋白也可用作ELISA和流式细胞术形式中EV定量的有力工具。因此，Tim4对EV的亲和力将在EV研究中找到丰富的应用。
Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+-dependent, intact EVs can be easily released from Tim4 by adding Ca2+ chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations’ proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies.
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DOI：
10.1073/pnas.1521230113
发表时间：
2016-02-23
影响因子：
11.1
作者：
Kowal, Joanna;Arras, Guillaume;Thery, Clotilde
通讯作者：
Thery, Clotilde
影响因子：
14.9
作者：
Ritchie ME;Phipson B;Wu D;Hu Y;Law CW;Shi W;Smyth GK
通讯作者：
Smyth GK
影响因子：
3.7
作者：
Ghosh A;Davey M;Chute IC;Griffiths SG;Lewis S;Chacko S;Barnett D;Crapoulet N;Fournier S;Joy A;Caissie MC;Ferguson AD;Daigle M;Meli MV;Lewis SM;Ouellette RJ
通讯作者：
Ouellette RJ
影响因子：
12.3
作者：
Gentleman RC;Carey VJ;Bates DM;Bolstad B;Dettling M;Dudoit S;Ellis B;Gautier L;Ge Y;Gentry J;Hornik K;Hothorn T;Huber W;Iacus S;Irizarry R;Leisch F;Li C;Maechler M;Rossini AJ;Sawitzki G;Smith C;Smyth G;Tierney L;Yang JY;Zhang J
通讯作者：
Zhang J
影响因子：
4.8
作者：
Savina, A;Furl치n, M;Colombo, MI
通讯作者：
Colombo, MI