Proteomics Uncovers Enrichment Bias of Common Extracellular Vesicle Isolation Methods in HEK293T Cells

Extracellular vesicles (EVs) are membranous structures consisting of lipid bilayers that are released by most cell types and serve as important mediators of intercellular communication. The HEK293T cell line model has gained considerable attention from the scientific community, particularly in the fields of engineering and drug delivery. Nevertheless, there is a dearth of systematic comparisons of the most prevalent EV isolation methodologies for HEK293T in terms of recovery and specificity. In this study, common EV isolation workflows using either ultracentrifugation (UC), ultrafiltration (UF), polyethylene glycol precipitation (PEG), or EXODUS were compared in HEK293T cells and profiled using proteomics, which enabled the identification of almost 4000 proteins. Moreover, the observed large discrepancy between PEG and other methods can be attributed to the cofractionation of two EV populations that differed in density and protein composition, as revealed by isopycnic ultracentrifugation. Therefore, it was assumed that the cargo inside EVs is correlated with their density, which presents intrinsic heterogeneity in HEK293T EVs. This study aimed to facilitate the selection and implementation of an EV enrichment procedure for the HEK293T cell line and to discern heterogeneity in the HEK293T EV population.