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  • Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques.

Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques.

Proteomics (2008-05-21)
Annette Dreisbach, Andreas Otto, Dörte Becher, Elke Hammer, Alexander Teumer, Joost W Gouw, Michael Hecker, Uwe Völker
ABSTRACT

Bacillus subtilis has been developed as a model system for physiological proteomics. However, thus far these studies have mainly been limited to cytoplasmic, extracellular, and cell-wall attached proteins. Although being certainly important for cell physiology, the membrane protein fraction has not been studied in comparable depth due to inaccessibility by traditional 2-DE-based workflows and limitations in reliable quantification. In this study, we now compare the potential of stable isotope labeling with amino acids (SILAC) and (14)N/(15)N-labeling for the analysis of bacterial membrane fractions in physiology-driven proteomic studies. Using adaptation of B. subtilis to amino acid (lysine) and glucose starvation as proof of principle scenarios, we show that both approaches provide similarly valuable data for the quantification of bacterial membrane proteins. Even if labeling with stable amino acids allows a more straightforward analysis of data, the (14)N/(15)N-labeling has some advantages in general such as labeling of all amino acids and thereby increasing the number of peptides for quantification. Both, SILAC as well as (14)N/(15)N-labeling are compatible with 2-DE, 2-D LC-MS/MS, and GeLC-MS/MS and thus will allow comprehensive simultaneous interrogation of cytoplasmic and enriched membrane proteomes.

MATERIALS
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Sigma-Aldrich
Ammonium-15N2 sulfate, 10 atom % 15N

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