Automated Proteomics Sample Preparation of Phosphatidylserine-Positive Extracellular Vesicles from Human Body Fluids

[Image: see text] Extracellular vesicles (EVs) are ubiquitously secreted by almost every cell type and are present in all body fluids. Blood-derived EVs can be used as a promising source for biomarker monitoring in disease. EV proteomics is currently being analyzed in clinical specimens. However, their EV proteomics preparation methods are limited in throughput for human subjects. Here, we introduced a novel automated EV isolation and sample preparation method using a magnetic particle processing robot for automated 96-well processing of magnetic particles for EV proteomics analysis that can be started with a low volume of multiple clinical samples. The automation of EV purification reduced the coefficient of variation of protein quantification from 3.5 to 2.2% compared with manual purification, enabling the quantification of 1120 proteins in 1 h of MS analysis. This automated proteomics EV sample preparation is attractive for processing large cohort samples for biomarker development, validation, and routine testing.