In-depth analysis of the plasma proteome: Are we enriching extracellular vesicles, platelets, or soluble proteins?

Plasma is a complex milieu comprising extracellular vesicles (EVs), residual platelets, and soluble proteins, each carrying information about physiological and pathological states. Traditional neat plasma analysis yields identification of hundreds of proteins, but recent strategies allow to expand coverage to thousands of proteins. These strategies primarily utilize centrifugation and magnetic nanoparticles. In this study, we compared three different strategies for achieving an in-depth analysis of plasma proteomics to evaluate the contribution of soluble proteins, EV cargo proteins, and platelets. We compared the protein profiles obtained from EV preparations isolated by centrifugation and enriched plasma fractions isolated using magnetic nanoparticle-based and beads methods.

We analyzed platelet-poor plasma (PPP) samples from five healthy donors. For each sample, we conducted neat plasma analysis and nanoparticle-based enrichment using the XT kit on the Proteograph assay (Seer), as well SCX magnetic beads (MagNet ResynBio) and multiple centrifugation steps to enrich the EV. Proteins were digested using STRAP and analyzed on the Evosep One coupled to a timsTOF HT (Bruker Datonics) for the MagNet assay and on the nanoElute2-timsTOF PRO (Bruker Datonics) using packed emitter column (C18, 25 cm × 75 μm 1.6 μm) (IonOpticks). Data were analyzed using DIA-NN in library-free mode. We combined ORA, GSEA and EV-specific markers, to assess the presence of EV cargo proteins in different preparations, providing insights into the functional relevance of EVs in plasma.

Preliminary results indicate that although all three strategies allowed identification of 1200-4500 proteins, and that they were very distinguishable by PCA analysis. From the samples, we quantified ca. 4500 using EV preparation by centrifugation, ca. 4200 using Nanoparticles Seer and about 1200 proteins using SCX beads MagNET. Samples exhibited distinct signatures of EVs, platelets, and soluble proteins, with significant enrichment of EV cargo proteins in EV preparations compared to nanoparticle-enriched plasma fractions. EV markers, CD63 and CD9 were two times more abundant in EV preparation compared to Seer, and were not detected in neat plasma and in MagNET. CD81 was uniquely identified in EV preparations. Complement related proteins were not significantly enriched in any samples. Further data acquisitions and analysis are underway to confirm these findings and elucidate further the specific pattern of identified proteins in each sample preparation type.

This analysis provides insights into the composition of plasma proteome enriched using different strategies, which can guide the choice of biomarker discovery approach.