Improving the profile and reliability of cytoplasmic proteins from M. tuberculosis using biomimetic affinity chromatography coupled with LC-MS/MS analysis.

M. tuberculosis is the most successful intracellular pathogen and remains a major threat to human health. It is crucial to investigate the profile of cytoplasmic proteins from M. tuberculosis for pathogenesis, clinical markers, and protein vaccine development. In this study, six biomimetic affinity chromatography (BiAC) resins with high differences were selected for M. tuberculosis-cytoplasmic protein fractionation. All fractions were identified using liquid chromatography-mass spectrometry (LC-MS/MS) analysis. A total of 1246 M. tuberculosis proteins were detected (p < 0.05), among which 1092 M. tuberculosis proteins were identified in BiAC fractionations and 714 M. tuberculosis proteins in un-fractionations (Table S1.3.1). The majority of 66.8% (831/1246) identifications were distributed in the range of Mw 7.0-70.0 kDa, pI 3.5-8.0, and Gravy values <0.3. Furthermore, 560 M. tuberculosis proteins were detected in both the BiAC fractionations and un-fractionations. Compared with the un-fractionations, the average number of prot_matches, prot_cover, prot_sequence, and emPAI values of these 560 proteins in the BiAC fractionations were increased by 3.791, 1.420, 1.307, and 1.788 times, respectively. Overall, compared with un-fractionations, the confidence and profile of M. tuberculosis cytoplasmic proteins were improved by BiAC fractionations coupled with LC-MS/MS analysis. The strategy of BiAC fractionation can be used as an effective method for pre-separating protein mixtures in proteomic studies.