Hands-free Proteomic Profiling of Urinary Extracellular Vesicles with a High-throughput Automated Workflow

Extracellular vesicles (EVs) have emerged as a promising source of disease biomarkers for non-invasive early-stage diagnoses, but a bottleneck in EV sample processing restricts their immense potential in clinical applications. Existing methods are limited by low EV yield and integrity, slow processing speeds, low sample capacity, and poor recovery efficiency. We aimed to address these issues with a high-throughput, automated workflow for EV isolation, EV lysis, protein extraction, and protein denaturation. The automation can process clinical urine samples in parallel, resulting in protein-covered beads ready for various analytical methods, including immunoassays, protein quantitation assays, and mass spectrometry. Compared to the standar­­­d manual lysis method for contamination levels, efficiency, and consistency of EV isolation, the automated protocol shows reproducible and robust proteomic quantitation with less than 10% median coefficient of variation. When we applied the method to clinical samples, we identified a total 3,793 unique proteins and 40,380 unique peptides, with 992 significantly upregulated proteins in kidney cancer patients versus healthy controls. These upregulated proteins were found to be involved in several important kidney cancer metabolic pathways also identified with a manual control. This hands-free workflow represents a practical EV extraction and profiling approach that can benefit both clinical and research applications, streamlining biomarker discovery, tumor monitoring, and early cancer diagnoses.