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 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.
Twine
Susan M
SM
Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario, Canada. Susan.Twine@nrc-cnrc.gc.ca
Mykytczuk
Nadia C S
NC
Petit
Mireille
M
Tremblay
Tammy-Lynn
TL
Lanthier
P
P
Conlan
J Wayne
JW
Kelly
John F
JF
D001426:Bacterial Proteins / Q000737:chemistry; D001622:Betaine / Q000031:analogs & derivatives / Q000494:pharmacology; D002254:Carbonates / Q000737:chemistry / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D005604:Francisella tularensis / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D007525:Isoelectric Focusing; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D013890:Thiourea / Q000494:pharmacology; D014357:Trypsin / Q000737:chemistry; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D002254:Carbonates; D002793:Cholic Acids; D003902:Detergents; D011506:Proteins; C506587:amidosulfobetaine-14; D001622:Betaine; C005686:sodium carbonate; C483727:sulfobetaine; D014508:Urea; D014357:Trypsin; D013890:Thiourea; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D004229:Dithiothreitol