Optimizing Size Exclusion Chromatography for Extracellular Vesicle Enrichment and Proteomic Analysis from Clinically...

The field of extracellular vesicle (EV) research has rapidly expanded in recent years, with particular interest in their potential as circulating biomarkers. Proteomic analysis of EVs from clinical samples is complicated by the low abundance of EV proteins relative to highly abundant circulating proteins such as albumin and apolipoproteins. To overcome this, size exclusion chromatography (SEC) has been proposed as a method to enrich EVs whilst depleting protein contaminants; however, the optimal SEC parameters for EV proteomics have not been thoroughly investigated. Here, quantitative evaluation and optimization of SEC are reported for separating EVs from contaminating proteins. Using a synthetic model system followed by cell line-derived EVs, it is found that a 10 mL Sepharose 4B column in PBS produces optimal resolution of EVs from background protein. By spiking-in cancer cell-derived EVs to healthy plasma, it is shown that some cancer EV-associated proteins are detectable by nano-LC-MS/MS when as little as 1% of the total plasma EV number are derived from a cancer cell line. These results suggest that an optimized SEC and nanoLC-MS/MS workflow may be sufficiently sensitive for disease EV protein biomarker discovery from patient-derived clinical samples.