High-EfficiencyCapture and Proteomic Analysis ofPlasma-Derived Extracellular Vesicles through Affinity Purification

Plasma-derived extracellular vesicles (EVs) are promising sources of biomarkers. It is still a challenge to isolate EVs from a small amount of human plasma for downstream proteomic analysis. The isolation process is hindered by contamination with high-abundance blood proteins and lipoprotein particles, which adversely impact proteomic analyses. Moreover, although EV immune-isolation via magnetic beads often integrates with flow sorting and Western blotting (WB), it lacks compatibility with nanoparticle tracking analysis (NTA) and proteomic analysis. To address these issues, we have developed a functional affinity magnetic bead, EVlent (Extracellular Vesicles isoLated Efficiently, Naturally, and Totally), enabling the rapid and efficient isolation of EVs from plasma. By optimizing the quantities of magnetic beads and plasma used, we characterized the isolated EVs through WB, NTA, and transmission electron microscopy (TEM), showing the successful isolation of EVs from plasma. Proteomic analysis of these EVs identified over 2000 proteins and 15,000 peptides from 100 μL of plasma and nearly 1000 proteins from trace samples as small as 5 μL. Additionally, this isolation method significantly reduced contaminants, including plasma proteins and lipoproteins, compared to ultracentrifugation. Finally, we applied this strategy to plasma samples of healthy individuals and those with Parkinson’s disease, identifying four potential biomarkers that provide promising guidance for clinical diagnosis.