Abstract Extracellular vesicles (EVs) in blood plasma offer a valuable reservoir of intracellular cellular cargo, making them a promising source of liquid based biomarkers. The molecular cargo of small EVs (sEVs) is of particular interest because some EV subtypes encapsulate cargo from organelles including mitochondria, endosomes, and the autophagy pathways, which are implicated in multiple diseases. However, the complexity of plasma, with its abundance of non-EV particles and plasma proteins, presents challenges for their molecular characterization using mass spectrometry based ‘omics technologies. Here, we optimised a rigorous method to isolate sEVs from human plasma based on both density and size. Following this, we analysed the protein and lipid content of sEVs from multiple individuals. We demonstrate the advantage of obtaining highly enriched sEVs from plasma for enhancing the detection of protein networks associated with mitochondria and the endosomal network, and also tissue types including the central nervous system. Some of the EV associated proteins reported here have not been detected in plasma, nor plasma sEVs, previously. We show that sphingomyelin lipids are the most abundant lipids in plasma sEVs (33.7 mol% total lipids) and provide the first report on cholesterol ester content. We demonstrate a 16-fold decrease in cholesterol ester lipids in sEVs compared to platelet free plasma and suggest that cholesterol ester content could serve as a valuable measure for assessing the effectiveness of plasma separation protocols or kits in enriching for sEVs. Our study highlights the benefit of reducing co-isolates from plasma sEV preparations to enable the detection of proteins and lipids with potential biomarker utility, and underscores the need for ongoing development of improved high throughput sEV isolation technologies.

Multi-omics characterization of highly enriched human plasma extracellular vesicles.