Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography.
Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography.
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DOI：
10.1002/jev2.12087
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发表时间：
2021-04
影响因子：
16
通讯作者：
Martínez-Martínez E
中科院分区：
文献类型：
--
作者：
Martínez-Greene JA;Hernández-Ortega K;Quiroz-Baez R;Resendis-Antonio O;Pichardo-Casas I;Sinclair DA;Budnik B;Hidalgo-Miranda A;Uribe-Querol E;Ramos-Godínez MDP;Martínez-Martínez E
细胞外囊泡（EV）的分子表征揭示了其组成在细胞和组织水平上的巨大异质性。目前的分离方法不能有效地分离EV亚型用于蛋白质组学和功能分析。本研究的目的是开发一种可重复和可扩展的分离工作流程，以提高EV制剂的产率和纯度。通过基于聚合物的沉淀和尺寸排阻色谱（Pre‐SEC）的组合，我们基于其CD 9、CD 63和CD 81含量和洗脱时间分析了EV的两个子集。使用透射电子显微镜、纳米颗粒跟踪分析和蛋白质印迹分析来表征EV。为了评价早期和晚期洗脱EV组分之间蛋白质组成的差异，我们对MDA-MB-468衍生EV进行了定量蛋白质组学分析。我们在早期洗脱组分中鉴定了286种排他性蛋白质，并在早期和晚期洗脱组分之间鉴定了148种具有差异浓度的蛋白质。密度梯度分析进一步揭示了每个分析亚组内的EV异质性。通过系统生物学的方法，我们发现在EV中包含的蛋白质之间的显着的相互作用，这表明存在与特定的生物过程相关的功能簇。这里介绍的工作流程允许在单个细胞类型内研究EV亚型，并有助于标准化功能研究的EV分离。
The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer‐based precipitation and size exclusion chromatography (Pre‐SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early‐ and late‐eluting EV fractions, we performed a quantitative proteomic analysis of MDA‐MB‐468‐derived EVs. We identified 286 exclusive proteins in early‐eluting fractions and 148 proteins with a differential concentration between early‐ and late‐eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional 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：
3.7
作者：
Duong, Phat;Chung, Allen;Raffai, Robert L.
通讯作者：
Raffai, Robert L.
影响因子：
5.2
作者：
Libring, Sarah;Shinde, Aparna;Solorio, Luis
通讯作者：
Solorio, Luis
影响因子：
3.4
作者：
Lane, Rebecca E.;Korbie, Darren;Hill, Michelle M.
通讯作者：
Hill, Michelle M.
影响因子：
3.4
作者：
Anand, Sushma;Foot, Natalie;Kumar, Sharad
通讯作者：
Kumar, Sharad