Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques.
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.
Dreisbach
Annette
A
Interfakultäres Institut für Genetik und Funktionelle Genomforschung, Ernst-Moritz-Arndt-Universität, Greifswald, Germany.
Otto
Andreas
A
Becher
Dörte
D
Hammer
Elke
E
Teumer
Alexander
A
Gouw
Joost W
JW
Hecker
Michael
M
Völker
Uwe
U
D000222:Adaptation, Physiological; D001412:Bacillus subtilis / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D015964:Gene Expression Regulation, Bacterial; D007553:Isotope Labeling / Q000379:methods; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
D001426:Bacterial Proteins; D008565:Membrane Proteins