Suresh Patil Shivprasad, Zhang Qibin
Center for Translational Biomedical Research, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, North Carolina 28081, United States.
Department of Chemistry & Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina 27402, United States.
J Proteome Res. 2025 Jun 6;24(6):2956-2967. doi: 10.1021/acs.jproteome.5c00149. Epub 2025 May 12.
Extracellular vesicles (EVs) are a vital component in cell-cell communication and hold significant potential as biomarkers and therapeutic carriers. Having a reproducible and simple EV isolation method for small volumes of human plasma is essential for biomarker discovery. Although combining multiple methods has been a recent trend in its ability to minimize contamination, it is not ideal for clinical specimens due to the large sample number and small sample volume. This study compared EVs isolated from 100 μL of plasma by nine commonly used methods based on different principles, including centrifugation, polymer precipitation, size exclusion, electrostatic interaction, and affinity enrichment. The isolated EVs were characterized by particle size and number using nanoparticle tracking analysis, purity, and contaminants using Simple Western and overall proteomic profiles using bottom-up proteomics. Despite the same EV enrichment principle, individual methods isolated EVs exhibited distinct characteristics, likely due to variations in the physicochemical properties of materials used and specific protocols. Overall, all of the methods evaluated are reproducible. MagNet and MagCap methods result in purer EVs with the narrowest size distribution and the highest proteome coverage but modest yield. This is the first report on isolating EVs from 100 μL of plasma using nine different methods with detailed characterization.
细胞外囊泡（EVs）是细胞间通讯的重要组成部分，作为生物标志物和治疗载体具有巨大潜力。拥有一种可重复且简单的用于小体积人血浆的EV分离方法对于生物标志物的发现至关重要。尽管结合多种方法已成为近期减少污染能力的趋势，但由于临床标本数量大且样本体积小，这种方法并不理想。本研究比较了基于不同原理的九种常用方法从100μL血浆中分离的EVs，这些原理包括离心、聚合物沉淀、尺寸排阻、静电相互作用和亲和富集。使用纳米颗粒跟踪分析对分离的EVs进行粒径和数量表征，使用Simple Western对纯度和污染物进行分析，并使用自下而上的蛋白质组学对整体蛋白质组图谱进行分析。尽管EV富集原理相同，但个别方法分离的EVs表现出不同的特征，这可能是由于所用材料的物理化学性质和特定方案的差异所致。总体而言，所有评估的方法都是可重复的。MagNet和MagCap方法可产生纯度更高的EVs，其尺寸分布最窄，蛋白质组覆盖率最高，但产量适中。这是第一篇关于使用九种不同方法从100μL血浆中分离EVs并进行详细表征的报告。
