Optimizing Mass Spectrometry Methods to Uncover M. tuberculosis Biomarkers in Extracellular Vesicles

Individuals exposed to Mtb can experience a range of outcomes: early bacterial clearance, latent Mtb infection (LTBI), asymptomatic TB (bacteriologically confirmed or not), or symptomatic TB (bacteriologically confirmed or clinically diagnosed). Current tools cannot reliably predict or distinguish these stages. Previous studies have identified Mtb peptides in extracellular vesicles (EVs) from TB patients’ serum, highlighting their potential as biomarkers. We aimed to identify Mtb proteins and peptides in serum EVs as potential biomarkers across TB stages, with a focus on differentiating asymptomatic individuals. We evaluated data-independent acquisition (DIA) mass spectrometry with different data analysis strategies to identify and differentially quantify Mtb peptides. We analyzed serum samples from healthy, HIV-negative South African adult volunteers enrolled in a TB risk study. Samples were categorized as prevalent TB (diagnosed within 30 days of baseline), incident TB (collected at baseline from participants who later developed TB), activated TB (collected at diagnosis, during follow-up, from incident cases), or controls (collected at baseline from participants who did not develop TB within the 15 months of the study).