title: Phosphoproteomic analysis of bacillus Calmette-Guérin using gel-based and gel-free approaches.
abstract: Post-translational modifications regulate many aspects of protein behavior and provide options for expanding protein functionality in organisms. Protein phosphorylation is one of the major PTMs observed in bacteria, which are involved in regulating a myriad of physiological processes. Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been recognized as an important weapon in the fight against tuberculosis (TB) worldwide for over 80 years. In this study, we conducted phosphoproteomic analysis in BCG bacteria using gel-based and gel-free complementary approaches and high-resolution Fourier transform mass spectrometry. In total, 501 phosphopeptides derived from 398 phosphoproteins were identified, representing the first phosphoproteomic analysis of BCG reported to date. Thirty-three novel protein products supported by 36 unique phosphorylated peptides were detected. Additionally, the translational start sites of 28 proteins were confirmed, and 31 proteins were validated through the extension of translational start sites based on N-terminus-derived peptides. The expression of three randomly selected phosphoproteins was validated through Western blotting. A number of proteins involved in metabolic pathways, including glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation and two-component system, are discussed. We believe some of the proteins identified in this study may represent potential targets for the development of novel antibiotics for treating TB.
authors.affiliation: MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zheng
authors.affiliation: MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Liguo
authors.initials: L
authors.lastname: Liu
authors.affiliation: MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Liu
authors.affiliation: MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. Electronic address: zdsys@vip.sina.com.
authors.forename: Qi
authors.initials: Q
authors.lastname: Jin
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D009163:Mycobacterium bovis / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D010750:Phosphoproteins
keywords: Metabolic pathway; Mycobacterium bovis bacillus Calmette–Guérin; Phosphoproteome; SDS-PAGE; TiO(2) enrichment