Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins

TLDR

Isolation of extracellular vesicles from blood is crucial for understanding their biology and developing disease biomarkers, yet concurrent lipoprotein particles make blood a challenging fluid for EV purification. The study aimed to develop a robust method to isolate and characterize blood EVs with minimal contamination by plasma proteins and lipoproteins. Researchers collected plasma and serum from healthy subjects and isolated EVs by size‑exclusion chromatography, then further purified them by adding a density cushion to SEC, which reduced lipoprotein contamination by 100‑fold. Using this combined SEC‑density cushion approach, 1,187 proteins were identified in plasma EVs, including previously unreported EV‑associated proteins, demonstrating that the method enables detailed proteomic analysis and establishes blood as a viable source for EV biomarker discovery.

Abstract

The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8–12, while the bulk of the plasma proteins was present in fractions 11–28. Vesicle markers peaked in fractions 7–11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.