Enhancing detection efficiency of RD-proteins in Mycobacterium tuberculosis culture filtrate by biomimetic affinity chromatography coupled with LC-MS/MS

Mycobacterium tuberculosis culture filtrate proteins (MTB-CFPs) are critical for tuberculosis diagnosis and vaccine development. To explore their diagnostic potential, MTB was statically cultured in Sauton's medium for 15 days; MTB-CFPs were collected, fractionated using six biomimetic affinity chromatography (BiAC) resins, and analyzed by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Four MTB-specific regions of difference (RDs) and the PstS1 protein were recombinantly expressed and purified, with their serological efficacy assessed by ELISA using sera from 157 pulmonary tuberculosis (pTB) patients and 71 healthy controls (HCs). LC-MS/MS identified 622 MTB proteins, 307 of which were validated with ≥2 peptides and P < 0.05. BiAC fractionation significantly increased the average peptide number (2.09-fold), peptide coverage (1.43-fold), and the exponentially modified Protein Abundance Index (emPAI) (1.70-fold) in 281 common identifications. Receiver Operating Characteristic (ROC) analysis revealed rEC (a fusion expression product of the Rv3875 and Rv3874 genes in the RD1 region, linked by a 15-nucleotide connector) as the optimal diagnostic marker Area Under the Curve (AUC = 0.8030), with sharply distinct IgG levels between pTB patients and HCs (P < 0.0001). Notably, rEC combined with GmdA (encoded by the Rv1511 gene located in RD11) enhanced sensitivity while preserving high specificity, especially for Acid-Fast Bacilli (AFB) negative pTB cases demonstrating substantial clinical translation potential. Additionally, extended culture promoted MTB autolysis and cytoplasmic protein release, providing valuable insights into TB pathogenesis and biomarker discovery. This integrated approach of BiAC fractionation, proteomics, and serological validation advances TB precision diagnostics.