Membrane and membrane-associated proteins in Triton X-114 extracts of Mycobacterium bovis BCG identified using a combination of gel-based and gel-free fractionation strategies.
Tuberculosis is an ancient disease that remains a significant global health problem. Because many membrane and membrane-associated proteins of this pathogen represent potential targets for drugs, diagnostic probes or vaccine components, we have analysed Mycobacterium bovis, bacillus Calmette-Guérin (BCG) substrain Moreau, using Triton X-114 for extraction of lipophilic proteins, followed by identification with LC coupled MS/MS. We identified 351 different proteins in total, and 103 (29%) were predicted as integral membrane proteins with at least one predicted transmembrane region and another 84 (23.9%) proteins had a positive grand average of hydropathicity (GRAVY) value, indicating increased probability for membrane association. Altogether 43 predicted lipoproteins (Lpps) were identified which is close to 50% of the total number of Lpps in the genome. Fifty-four proteins, including twenty-four predicted integral membrane proteins and seven predicted Lpps are described for the first time. The proportion of hydrophobic membrane and membrane-associated proteins shows that Triton X-114 is a highly efficient method for extraction of membrane proteins from bacteria, without the need for preisolation of membranes. ATP synthase, NAD(P) transhydrogenase, ubiquinone oxidoreductase and ubiquinol-cytochrome C reductase appear to represent major enzyme complexes in the membrane of Mycobacterium tuberculosis complex organisms.
Målen
Hiwa
H
Section for Microbiology and Immunology, The Gade Institute, University of Bergen, Bergen, Norway.
Berven
Frode S
FS
Søfteland
Tina
T
Arntzen
Magnus Øverlie
MØ
D'Santos
Clive S
CS
De Souza
Gustavo Antonio
GA
Wiker
Harald G
HG
D025181:ATP Synthetase Complexes / Q000378:metabolism; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D003902:Detergents / Q000494:pharmacology*; D042967:Electron Transport Complex I / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000737:chemistry; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D013347:Subcellular Fractions
D003902:Detergents; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D009250:NADP Transhydrogenases; D025181:ATP Synthetase Complexes; D042967:Electron Transport Complex I