Comparative Analysis of Platelet-Derived Extracellular Vesicle Protein Extraction Methodologies for Mass Spectrometry.

The aim of this study is to present a comparative study of different methodologies for the extraction of proteins from platelet-derived extracellular vesicles (pEVs) prior to subsequent mass spectrometry (MS) analysis. pEVs were isolated by size exclusion chromatography (SEC) from human platelet lysates (PL) and characterized by identifying specific markers by Western blot, visualizing morphology 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 proteins from pEV and could be considered according to their intrinsic characteristics, in accordance with the research requirements.