Multiomics analysis to evaluate the enrichment of extracellular vesicles from human plasma

Extracellular vesicles (EVs) in blood plasma offer a valuable reservoir of intracellular cellular cargo, making them a promising source of liquid-based biomarkers. However, the complexity of plasma, with its abundance of non-EV particles and plasma proteins, presents challenges for their molecular characterization, particular their lipid composition, using mass spectrometry-based technologies. Consequently, there is currently no comprehensive blueprint detailing both the proteomes and lipidomes of highly enriched plasma EVs. We employed an orthogonal approach using density gradient ultracentrifugation (DGUC) and size-exclusion chromatography (SEC) to isolate EVs and conducted a comparative study on four different SEC columns following DGUC to evaluate the capacity of the SEC columns in enriching EVs while depleting plasma proteins and lipoprotein particles. The EV fractions were analyzed with data-independent acquisition proteomics and nano-ESI-ultrahigh-resolution accurate mass spectrometric lipidomics. DGUC followed by the appropriated sized SEC provided the best enrichment of EVs and the corresponding depletion of plasma protein and lipoprotein particle contaminants. We show that glycerophosphoethanoamine, glycerophosphoserine, ceramide, and sphingomyelin lipids are significantly enriched, while cholesteryl ester content is significantly depleted in EVs compared to platelet depleted plasma. This strategy also enabled the detection of proteins in the enriched EV fractions with functions related to mitochondria, endosomal-autophagic-lysosomal pathways, and the central nervous system. This study highlights the benefit of depleting coisolates from plasma EV preparations to enable the detection of proteins and lipids with potential future clinical utility and underscores the need for ongoing development of improved high-throughput EV isolation technologies.