Session: Parallel session 10 - Health and biological sciences

In-depth Analysis of 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, all potentially informing pathological states. Direct plasma analysis yields to hundreds of proteins, but recent strategies expand coverage to thousands. We compared three strategies for in-depth plasma proteomics to evaluate soluble proteins, EV cargo proteins, and platelets, focusing on protein profiles from EV preparations isolated by centrifugation and enriched plasma fractions using nanoparticle-based methods.

 

We analyzed platelet-poor plasma (PPP) samples from five healthy donors. Each sample underwent neat plasma analysis, nanoparticle-based enrichment using the XT kit on the Proteograph assay (Seer), SCX magnetic beads (MagNet ResynBio), and multiple centrifugation steps to enrich EVs. Proteins were digested using STRAP and analyzed on the EvosepOne-timsTOF HT for the MagNet assay, and on the nanoElute2-timsTOF PRO for the rest. Data were analyzed using DIA-NN in library-free mode to quantify proteins in each preparation. We combined ORA, GSEA, and EV-specific markers to assess EV cargo proteins' presence, and to gain insights into enriched plasma composition.



From the same samples, we quantified ca.4500 proteins using EV preparation by centrifugation, ca.4200 using Nanoparticles Seer, and ca.1200 using MagNET. Samples exhibited distinct signatures of EVs, platelets, and soluble proteins.  We found significant enrichment of EV cargo proteins in EV preparations compared to nanoparticle-enriched plasma fractions. EV markers CD63 and CD9 were twice as abundant in EV preparations compared to Seer, and were absent in neat plasma and MagNET. CD81 was uniquely identified in EV preparation. Complement-related proteins were not significantly enriched in any samples. Further data acquisitions and analysis are underway to elucidate the composition of plasma proteins enriched using different strategies, informing biomarker discovery application choice.