Optimized Mass Spectrometry to Uncover <i>M. tuberculosis</i> Biomarkers in Extracellular Vesicles From Asymptomatic Tuberculosis Patients

BACKGROUND: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of death from a single infectious agent worldwide. Exposure to Mtb results in diverse outcomes: bacterial clearance, latent infection, asymptomatic, or symptomatic TB. Current diagnostic tools cannot reliably distinguish these outcomes. Our previous studies identified Mtb proteins in extracellular vesicles (EVs) from TB patients' serum, suggesting their potential as biomarkers. Here, we aimed to discover Mtb proteins and peptides in serum EVs across TB stages, focusing on asymptomatic individuals. METHODS: Serum was obtained from healthy, HIV-negative South African adult volunteers enrolled in a TB risk study. Based on patients' outcomes, samples were classified as prevalent, incident (controls that progressed to TB), activated TB, or community controls. EVs were isolated using ExoQuick™, followed by protein digestion and mass spectrometry (MS) analysis. Data-independent acquisition (DIA) with five different data analysis strategies, and two data-dependent (DDA) methods were used to identify Mtb proteins. RESULTS: Our DIA analysis using ion-mobility and spectral libraries enriched with Mtb-MS data revealed 19 Mtb proteins. Rv2997 was significantly higher at baseline in individuals who were initially TB-negative (incident) but later became bacteriologically positive, asymptomatic-TB (activated). HspX, GroEL2, and GroES, together with a MtrB peptide were significantly different between controls and asymptomatic-TB cases. DDA approaches did not resolve additional Mtb proteins. CONCLUSIONS: Quantitative DIA analysis discovered Mtb proteins/peptides in serum-derived EVs that were differentially abundant in individuals with early, asymptomatic TB. These findings highlight their potential as biomarkers and provide insight into host-pathogen interactions during subclinical infection.