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

Abstract

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.

Keywords: extracellular vesicles; mass spectrometry; methodologies; platelets; proteins; proteomics.

1

Graphical representation of the different methodologies used for protein extraction from pEV samples.

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pEV’s characterization by Western blot and TEM. (A) Representative images obtained by transmission electron microscopy (TEM) for pEV samples acquired at 94,000× magnification and 80 kV. (B) Identification of characteristic markers of EVs by Western blot. Tetraspanins CD9 and CD63, cytosolic HSC70, and coisolated albumin were identified in the different pEV samples, as well as in their respective original samples (PL). pEV1, pEV2, and pEV3 constitute three biological replicates, each obtained from a different platelet lysate pool of 50 donors.

3

Results of the proteomic analysis related to the comparative methodology. (A) Principal component analysis (PCA) applied to the data set. The clustering areas associated with the SDS-PAGE, OSP, and MB methodologies are represented in red, green, and blue, respectively. (B) Normalized protein abundances. Diagram was generated by calculating the arithmetic means of the normalized abundances of each protein detected in the different samples, considering each methodology. Results were statistically compared with Kruskal–Wallis’ test. (C) Venn diagram depicting the number of common and unique proteins related to each methodology.

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Bioinformatic analysis of protein data set. (A) Venn diagram representing the number of proteins identified under each methodology and listed in the Vesiclepedia database under “human” and “protein” terms. (B) Common proteins identified under each methodology and listed in the Vesiclepedia database Top 100 EV markers. Enrichment analysis of (C) cellular components and (D) site of expression categories using FunRich database as background.