Proteomics, phosphoproteomics and miRNA analysis of circulating extracellular vesicles through automated and high-throughput isolation

Here, we introduce a magnetic bead-based EV enrichment approach (EVrich) for automated and high-throughput processing of urine samples. Parallel enrichments can be performed in 96-well plates for downstream cargo analysis, including EV characterization, miRNA, proteomics and phosphoproteomics analysis. We applied the instrument to a cohort of clinical urine samples to achieve reproducible identification of over 47,562 unique EV peptides and 4,327 EV proteins in each one-ml urine sample. Quantitative phosphoproteomicsrevealed 186 unique phosphopeptides corresponding to 77 proteins that were significantly elevated in prostate cancer patients.