Platelet Proteomics Group, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade Santiago de Compostela, Santiago de Compostela, 15782, Spain.
Instituto de Investigación, Sanitaria de Santiago (IDIS), Santiago de Compostela, 15706, Spain.
Proteomics. 2019 Jan;19(1-2):e1800247. doi: 10.1002/pmic.201800247. Epub 2019 Jan 7.
Extracellular vesicles (EVs) are a heterogeneous population of vesicles composed of a lipid bilayer that carry a large repertoire of molecules including proteins, lipids, and nucleic acids. In this review, some guidelines for plasma-derived EVs isolation, characterization, and proteomic analysis, and the application of the above to cardiovascular disease (CVD) studies are provided. For EVs analysis, blood samples should be collected using a 21-gauge needle, preferably in citrate tubes, and plasma stored for up to 1 year at -80°, using a single freeze-thaw cycle. For proteomic applications, differential centrifugation (including ultracentrifugation steps) is a good option for EVs isolation. EVs characterization is done by transmission electron microscopy, particle enumeration techniques (nanoparticle-tracking analysis, dynamic light scattering), and flow cytometry. Regarding the proteomics strategy, a label-free and gel-free quantitative method is a good choice due to its accuracy and because it minimizes the amount of sample required for clinical applications. Besides the above, main EVs proteomic findings in cardiovascular-related diseases are presented and analyzed in this review, paying especial attention to overlapping results between studies. The latter might offer new insights into the clinical relevance and potential of novel EVs biomarkers identified to date in the context of CVD.
细胞外囊泡（EVs）是由脂质双层组成的异质囊泡群体，携带包括蛋白质、脂质和核酸在内的大量分子。在这篇综述中，提供了一些关于血浆衍生 EVs 分离、表征和蛋白质组学分析的指南，以及将上述方法应用于心血管疾病（CVD）研究的指南。对于 EVs 分析，应使用 21 号针头采集血液样本，最好使用柠檬酸盐管，并在 -80°C 下储存长达 1 年，最多可进行一次冻融循环。对于蛋白质组学应用，差速离心（包括超速离心步骤）是 EVs 分离的一个很好的选择。EVs 的表征通过透射电子显微镜、颗粒计数技术（纳米颗粒跟踪分析、动态光散射）和流式细胞术进行。关于蛋白质组学策略，由于其准确性以及可以最大限度地减少临床应用所需的样品量，因此无标记和无胶定量方法是一个不错的选择。除此之外，本文还介绍和分析了与心血管疾病相关的疾病中 EVs 的主要蛋白质组学发现，特别关注研究之间的重叠结果。后者可能为迄今为止在 CVD 背景下鉴定的新型 EVs 生物标志物的临床相关性和潜在性提供新的见解。
