Establishment of Minimum Protein Standards for Mycobacterium tuberculosis-Derived Extracellular Vesicles through Comparison of EV Enrichment Methods

Extracellular vesicles (EVs) produced by Mycobacterium tuberculosis, the causative agent of tuberculosis, are involved in acquiring nutrients, evading host immune mechanisms, and modulating the host immune response. The functions of these M. tuberculosis EVs (MEVs)are hypothesized to be due to the dynamic composition of MEVs. However, there are various methods used to purify MEVs, and these methods can impact MEV characteristics such as size, purity, integrity, and composition. In this study, we investigated several methods for MEV purification, including: ultracentrifugation, differential density gradient-based ultracentrifugation, qEV size exclusion chromatography, and Capto(TM)Core size exclusion chromatography. We evaluated the size, concentration, appearance, purity, and protein composition (by mass spectrometry) of MEVs from each of these methods. We ultimately found that protein and particle yield vary across methods and that the size exclusion chromatography-based methods are more technically reproducible than the ultracentrifugation-based methods. Through mass spectrometry analysis, we also identified seven M. tuberculosis proteins that were present regardless of MEV enrichment method, that we propose could be used as qualitative markers of MEVs versus contaminants (in addition to MEV size and appearance criteria), to benefit the reproducibility of ongoing MEV studies.