Stable Isotope Labeling by Amino Acids Applied to Bacterial Cell Culture

  • Boumediene Soufi
  • Boris MacekEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1188)


Stable isotope labeling by amino acids in cell culture (SILAC) is a widely used approach in quantitative proteomics; however, due to limitations such as required auxotrophy for the amino acids employed for labeling, it was thus far rarely employed in bacteria. Although limitations of SILAC in microbiological applications are significant and restrict its use exclusively to cells cultured in minimal media, we and others have successfully used it to fully label proteomes of model bacteria and measure their relative expression dynamics under different experimental conditions. Here we provide a brief overview of applications of SILAC in bacteria and describe a detailed protocol for SILAC labeling of Escherichia coli and Bacillus subtilis cells in culture, which in many cases can be applied to other members of both gram-positive and gram-negative bacterial species.

Key words

SILAC Quantitative proteomics Mass spectrometry Bacteria Prokaryotes 



The authors wish to thank Alejandro Carpy and the other PCT members for useful comments on the manuscript. Our work is financed by the Juniorprofessoren-Programm of the Landesstiftung BW, the SFB766 of the Deutsche Forschungsgemeinschaft, and PRIME-XS consortium.


  1. 1.
    Seraphin B, Hettich R (2012) Microbial proteomics: the quiet revolution. Curr Opin Microbiol 15(3):348–350PubMedCrossRefGoogle Scholar
  2. 2.
    Iwasaki M, Miwa S, Ikegami T et al (2010) One-dimensional capillary liquid chromatographic separation coupled with tandem mass spectrometry unveils the Escherichia coli proteome on a microarray scale. Anal Chem 82(7):2616–2620PubMedCrossRefGoogle Scholar
  3. 3.
    Otto A, Bernhardt J, Hecker M et al (2012) Global relative and absolute quantitation in microbial proteomics. Curr Opin Microbiol 15(3):364–372PubMedCrossRefGoogle Scholar
  4. 4.
    Ong SE, Mann M (2005) Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol 1(5):252–262PubMedCrossRefGoogle Scholar
  5. 5.
    Bantscheff M, Schirle M, Sweetman G et al (2007) Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem 389(4):1017–1031PubMedCrossRefGoogle Scholar
  6. 6.
    Macek B, Mann M, Olsen JV (2009) Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol 49:199–221PubMedCrossRefGoogle Scholar
  7. 7.
    Dreisbach A, Otto A, Becher D et al (2008) Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques. Proteomics 8(10):2062–2076PubMedCrossRefGoogle Scholar
  8. 8.
    Otto A, Bernhardt J, Meyer H et al (2010) Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis. Nat Commun 1:137PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Schutz W, Hausmann N, Krug K et al (2011) Extending SILAC to proteomics of plant cell lines. Plant Cell 23(5):1701–1705PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Soufi B, Kumar C, Gnad F et al (2010) Stable isotope labeling by amino acids in cell culture (SILAC) applied to quantitative proteomics of Bacillus subtilis. J Proteome Res 9(7):3638–3646PubMedCrossRefGoogle Scholar
  11. 11.
    Geiger T, Wisniewski JR, Cox J et al (2011) Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics. Nat Protoc 6(2):147–157PubMedCrossRefGoogle Scholar
  12. 12.
    Schwanhausser B, Gossen M, Dittmar G et al (2009) Global analysis of cellular protein translation by pulsed SILAC. Proteomics 9(1):205–209PubMedCrossRefGoogle Scholar
  13. 13.
    Doherty MK, Hammond DE, Clague MJ et al (2009) Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC. J Proteome Res 8(1):104–112PubMedCrossRefGoogle Scholar
  14. 14.
    Bendall SC, Hughes C, Stewart MH et al (2008) Prevention of amino acid conversion in SILAC experiments with embryonic stem cells. Mol Cell Proteomics 7(9):1587–1597PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Lossner C, Warnken U, Pscherer A et al (2011) Preventing arginine-to-proline conversion in a cell-line-independent manner during cell cultivation under stable isotope labeling by amino acids in cell culture (SILAC) conditions. Anal Biochem 412(1):123–125PubMedCrossRefGoogle Scholar
  16. 16.
    Frohlich F, Christiano R, Walther TC (2013) Native SILAC: metabolic labeling of proteins in prototroph microorganisms based on lysine synthesis regulation. Mol Cell Proteomics 12(7):1995–2005PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372PubMedCrossRefGoogle Scholar
  18. 18.
    Cox J, Matic I, Hilger M et al (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc 4(5):698–705PubMedCrossRefGoogle Scholar
  19. 19.
    Cox J, Neuhauser N, Michalski A et al (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10(4):1794–1805PubMedCrossRefGoogle Scholar
  20. 20.
    Stulke J, Hanschke R, Hecker M (1993) Temporal activation of beta-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool. J Gen Microbiol 139(9):2041–2045PubMedCrossRefGoogle Scholar
  21. 21.
    Kirchner M, Selbach M (2012) In vivo quantitative proteome profiling: planning and evaluation of SILAC experiments. Methods Mol Biol 893:175–199PubMedCrossRefGoogle Scholar
  22. 22.
    Tyanova S, Mann M, Cox J (2014) MaxQuant for In-Depth Analysis of Large SILAC Datasets. In:Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC), Warscheid B (ed) 1188: 351–364Google Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Proteome Center TuebingenUniversity of TuebingenTuebingenGermany

Personalised recommendations