Optimized enrichment of circulating extracellular vesicles from whole blood samples using PROSPR

Abstract

Aim: Whole blood constitutes a robust and readily available resource for the identification and characterization of systemically circulating substances. Despite the relevant number of blood specimens archived in biobanks globally, their utilization in extracellular vesicle (EV) research remains considerably underexploited. In this study, we present an optimized PRotein Organic Solvent PRecipitation (PROSPR) workflow, specifically designed for the rapid and standardized enrichment of EVs directly from untreated blood samples.

Methods: PROSPR workflow was applied for EV enrichment directly from untreated blood. Comprehensive biophysical characterization was performed using dedicated flow cytometry, tunable resistive pulse sensing, and transmission electron microscopy. In parallel, discovery-driven lipidomics and high-depth proteomics were applied to assess the suitability of PROSPR-derived EVs for advanced systems biology and multi-omics integration.

Results: PROSPR consistently yielded highly purified EV preparations that preserved canonical vesicular morphology, structural integrity, and stable ζ-potentials, indicative of intact membrane characteristics. Multi-omics analyses revealed distinct and coordinated lipid-protein signatures uniquely enriched in EV populations derived from whole blood. These signatures predominantly reflected platelet- and mitochondria-associated cargo that was not detectable in corresponding plasma-derived EV fractions, highlighting key molecular features missed by plasma-based EV profiling approaches.

Conclusion: Consequently, the optimized PROSPR workflow presented here offers a robust and accessible methodological strategy to fully utilize dormant, archived blood resources within global and national biobanks, thereby enabling comprehensive mechanistic insights and facilitating the discovery of novel diagnostic and prognostic circulating biomarkers.

Keywords

Extracellular vesicle enrichment / PROSPR workflow / whole-blood biobanking / untargeted lipidomics / high-depth proteomics / multi-omics integration / circulating biomarkers

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