Establishment of minimum protein standards for Mycobacterium tuberculosis-derived extracellular vesicles through comparison of EV enrichment methods.

Mycobacterium tuberculosis extracellular vesicles (MEV) have been described as having potent immunological activities that are both beneficial and harmful to the host. Key to understanding this conflicting information is the proteomic characterization of MEVs. However, there is neither a standard for a purification method nor markers to assess relative purity and quality of MEVs. In this study, we purified MEVs by four different methods (simple ultracentrifugation, differential density gradient-based ultracentrifugation, qEV size exclusion chromatography, and Capto™Core size exclusion chromatography) and assessed the variability of MEV characteristics (size, concentration, appearance, purity, and protein content) amongst isolation methods. The vesicle appearance and size were consistent across all methods; however variability was found between and within all methods, with simple ultracentrifugation demonstrating the most variability both in reproducibility and purity. Protein concentration and content, and particle yield and purity, varied amongst all methods. The two size exclusion chromatography-based methods were more technically reproducible than either ultracentrifugation-based method, while qEV size exclusion chromatography and differential density gradient ultracentrifugation afforded MEV samples of the highest purity. Nonetheless, all methods had 7 proteins in common, the Sec-independent membrane bound twin-arginine translocase TatA (Rv2094c), the periplasmic phosphate-binding lipoprotein PstS3 (Rv0928), the heparin binding hemagglutinin HBHA (Rv0475), lipoprotein antigens LprG (Rv1411c) and LpqH (Rv3763), a member of the conserved 13E12 repeat protein family P95201 (Rv0393), and the tuberculin related peptide Rv0431 (P96277), suggesting the use of these proteins as qualitative markers of MEVs versus contaminants, in addition to size and appearance criteria, to benefit reproducibility and consensus for ongoing MEV studies.

Supplementary information: The online version contains supplementary material available at 10.1186/s44350-025-00003-8.