Stable isotope labeling by amino acids in cell culture (SILAC) applied to quantitative proteomics of Bacillus subtilis.
We applied stable isotope labeling by amino acids in cell culture (SILAC) to large-scale quantitative proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic for lysine and high accuracy mass spectrometry for downstream analysis, we identified and quantified changes in the levels of more than 1500 proteins in each of the tested conditions with high biological and technical reproducibility. With a total of 1928 identified proteins, this study presents one of the most comprehensive quantitative proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under phosphate starvation, demonstrating the full compatibility of the method with site-specific detection and quantitation of phosphorylation events in bacteria.
Max Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Boumediene
B
Soufi
Chanchal
C
Kumar
Florian
F
Gnad
Matthias
M
Mann
Ivan
I
Mijakovic
Boris
B
Macek
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism; D018929:Cell Culture Techniques; D016000:Cluster Analysis; D007553:Isotope Labeling; D009587:Nitrogen Isotopes / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D013312:Stress, Physiological; D019802:Succinic Acid / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D020543:Proteome; D019802:Succinic Acid