Gel-free and gel-based proteomics in Bacillus subtilis: a comparative study.
The proteome of exponentially growing Bacillus subtilis cells was dissected by the implementation of shotgun proteomics and a semigel-based approach for a particular exploration of membrane proteins. The current number of 745 protein identifications that was gained by the use of two-dimensional gel electrophoresis could be increased by 473 additional proteins. Therefore, almost 50% of the 2500 genes expressed in growing B. subtilis cells have been demonstrated at the protein level. In terms of exploring cellular physiology and adaptation to environmental changes or stress, proteins showing an alteration in expression level are of primary interest. The large number of vegetative proteins identified by gel-based and gel-free approaches is a good starting point for comparative physiological investigations. For this reason a gel-free quantitation with the recently introduced iTRAQ (isobaric tagging for relative and absolute quantitation) reagent technique was performed to investigate the heat shock response in B. subtilis. A comparison with gel-based data showed that both techniques revealed a similar level of up-regulation for proteins belonging to well studied heat hock regulons (SigB, HrcA, and CtsR). However, additional datasets have been obtained by the gel-free approach indicating a strong heat sensitivity of specific enzymes involved in amino acid synthesis.
Wolff
Susanne
S
Institute for Microbiology, Ernst-Moritz-Arndt-Universität, D-17487 Greifswald, Germany.
Otto
Andreas
A
Albrecht
Dirk
D
Zeng
Jianru Stahl
JS
Büttner
Knut
K
Glückmann
Matthias
M
Hecker
Michael
M
Becher
Dörte
D
D001412:Bacillus subtilis / Q000378:metabolism* / Q000502:physiology*; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008565:Membrane Proteins; D011933:Reagent Kits, Diagnostic