Francisella tularensis proteome: low levels of ASB-14 facilitate the visualization of membrane proteins in total protein extracts

Proteomic analysis of bacterial pathogens isolated from in vivo sources, such as infected tissues, provides many challenges not the least of which is the limited quantity of sample available for analysis. It is, therefore, highly desirable to develop a one-step cellular lysis and protein solubilizat...

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Bibliographic Details
Published in:Journal of proteome research 2005-09, Vol.4 (5), p.1848-1854
Main Authors: Twine, Susan M, Mykytczuk, Nadia C S, Petit, Mireille, Tremblay, Tammy-Lynn, Lanthier, P, Conlan, J Wayne, Kelly, John F
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Betaine - analogs & derivatives
Betaine - pharmacology
Carbonates - chemistry
Carbonates - pharmacology
Cell Membrane - metabolism
Cholic Acids - pharmacology
Detergents - pharmacology
Dithiothreitol - pharmacology
Electrophoresis, Gel, Two-Dimensional
Francisella tularensis - metabolism
Hydrogen-Ion Concentration
Image Processing, Computer-Assisted
Isoelectric Focusing
Proteins - chemistry
Proteomics - methods
Thiourea - pharmacology
Trypsin - chemistry
Urea - pharmacology
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Summary:Proteomic analysis of bacterial pathogens isolated from in vivo sources, such as infected tissues, provides many challenges not the least of which is the limited quantity of sample available for analysis. It is, therefore, highly desirable to develop a one-step cellular lysis and protein solubilization method that minimizes protein losses and allows the maximum possible coverage of the proteome. Here, we have used standard sample buffer constituents including urea, thiourea and DTT, but varied the detergent composition of the buffers in order to achieve the best quality of gels and the greatest spot resolution. We found that the most efficient solubilizing solution in this case consisted of 7 M urea, 2 M thiourea, 1% DTT, 0.5% amidosulfobetaine-14 (ASB-14) and 4% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Inclusion of low levels of ASB-14 in solutions allowed visualization of a subset of 24 new protein spots in the Live Vaccine Strain (LVS) of Francisella tularensis and 21 spots in a virulent A-strain of the pathogen. Further investigation showed that 15 of the 24 enriched LVS spots were membrane or membrane-associated proteins suggesting that the optimized lysis and solubilization solution aids in the detection of more hydrophobic proteins. This methodology is now being applied to the analysis of Francisella obtained from in vivo sources.
ISSN:1535-3893
DOI:10.1021/pr050102u