title: Optimizing of a protein extraction method for Mycobacterium tuberculosis proteome analysis using mass spectrometry.
abstract: A critical step in proteomic analyses comprises the implementation of a reliable cell lysis method with high yields of qualitative proteins. In Mycobacteria, the protein extraction step is often hampered by the thick waxy cell wall which is rich in mycolic acids. Harsh disruption techniques to release proteins from the cells are thus required. Here, we demonstrate an optimized protein extraction procedure for Mycobacterium tuberculosis (Mbt) that results in protein extracts that are useful for all currently used proteomics platforms, including gel and LC-MS based strategies. We compared the effectiveness of using both thiourea and urea and/or SDS and DTT in the solubilization buffer, in combination or not with sonication and/or bead beating. After some preliminary optimization steps on fast-growing Mbt-like organisms, namely Mycobacterium smegmatis and Mycobacterium fortuitum, the final protein extraction protocol was tested on M. tuberculosis. Based on the concentrations of the proteins recovered from each of the tested methods and on the quality of the extracted proteins as evaluated by SDS PAGE, we propose a lysis buffer that contains both thiourea and urea, in combination with two mechanical cell disruption methods: sonication and bead beating. The optimized protocol results in protein extracts that are useful in M. tuberculosis proteomics studies based on any proteomics strategy or platform.
authors.affiliation: Institut Pasteur de Madagascar, Antananarivo, Madagascar. Electronic address: msylvianne@pasteur.mg.
authors.forename: Marie Sylvianne
authors.initials: MS
authors.lastname: Rabodoarivelo
authors.affiliation: Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Gent, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Aerts
authors.affiliation: Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Gent, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Vandamme
authors.affiliation: Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Gent, Belgium.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Palomino
authors.affiliation: Institut Pasteur de Madagascar, Antananarivo, Madagascar.
authors.forename: Voahangy
authors.initials: V
authors.lastname: Rasolofo
authors.affiliation: Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Gent, Belgium.
authors.forename: Anandi
authors.initials: A
authors.lastname: Martin
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000166:cytology; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D020543:Proteome
keywords: Mass spectrometry; Mycobacterium tuberculosis; Protein extraction; Proteomics