Comparative analysis of platelet-derived extracellular vesicles protein extraction methodologies for mass spectrometry

The purpose of this study is to present a comparative study of different methodologies for the extraction of platelet derived-extracellular vesicles (pEV) proteins prior to the subsequent mass spectrometry (MS) analysis. pEV were isolated by size exclusion chromatography (SEC) from human platelet lysates (hPL) and characterized by identifying specific markers by western blot, visualizing morphology and size by transmission electron microscopy (TEM) and analyzing concentration and size via nanoparticle tracking analysis (NTA). Protein isolation was performed through three different methodologies based on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), organic solvent precipitation (OSP) or magnetic beads (MB), followed by protein digestion and sample acquisition by LC-MS/MS. Clustering of the samples according to methodology is observed in the principal component analysis (PCA), although no significant differences in terms of normalized abundances are reached. Similarly, a small number of proteins were identified as unique by each methodology, with 91.3 % coincidence among all three procedures. In addition, the bioinformatic results of the enrichment analysis and the numbers of proteins already identified in the Vesiclepedia database are highly similar for the three methodologies. Overall, all three methodologies analyzed are optimal for the extraction of pEV-derived proteins and could be considered according to their intrinsic characteristics in accordance with the research requirements.

本研究旨在针对后续质谱（mass spectrometry，MS）分析前的血小板源性细胞外囊泡（platelet derived-extracellular vesicles，pEV）蛋白质提取方法开展对比研究。本研究通过尺寸排阻色谱法（size exclusion chromatography，SEC）从人血小板裂解液（human platelet lysates，hPL）中分离pEV，并通过蛋白质免疫印迹（western blot）鉴定特异性标志物、透射电子显微镜（transmission electron microscopy，TEM）观察其形态与粒径，以及纳米颗粒追踪分析（nanoparticle tracking analysis，NTA）分析其浓度与粒径。蛋白质提取分别采用基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-polyacrylamide gel electrophoresis，SDS-PAGE）、有机溶剂沉淀法（organic solvent precipitation，OSP）或磁珠法（magnetic beads，MB）的三种不同方案，随后进行蛋白质酶解，并通过液相色谱-串联质谱（LC-MS/MS）完成样本数据采集。主成分分析（principal component analysis，PCA）结果显示，样本可按提取方法进行聚类，但在标准化丰度层面未出现显著差异。同理，各方法仅能鉴定出少量特有蛋白质，三种方法的总蛋白重合度达91.3%。此外，三种方法的富集分析生物信息学结果，以及已在Vesiclepedia数据库中鉴定到的蛋白质数量均高度相似。综上，所评估的三种方法均适用于pEV源性蛋白质的提取，可根据研究需求结合其固有特性进行选用。