Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Seraphin B, Hettich R (2012) Microbial proteomics: the quiet revolution. Curr Opin Microbiol 15(3):348–350
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):2616–2620
Otto A, Bernhardt J, Hecker M et al (2012) Global relative and absolute quantitation in microbial proteomics. Curr Opin Microbiol 15(3):364–372
Ong SE, Mann M (2005) Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol 1(5):252–262
Bantscheff M, Schirle M, Sweetman G et al (2007) Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem 389(4):1017–1031
Macek B, Mann M, Olsen JV (2009) Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol 49:199–221
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):2062–2076
Otto A, Bernhardt J, Meyer H et al (2010) Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis. Nat Commun 1:137
Schutz W, Hausmann N, Krug K et al (2011) Extending SILAC to proteomics of plant cell lines. Plant Cell 23(5):1701–1705
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):3638–3646
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):147–157
Schwanhausser B, Gossen M, Dittmar G et al (2009) Global analysis of cellular protein translation by pulsed SILAC. Proteomics 9(1):205–209
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):104–112
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):1587–1597
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):123–125
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):1995–2005
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):1367–1372
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):698–705
Cox J, Neuhauser N, Michalski A et al (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10(4):1794–1805
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):2041–2045
Kirchner M, Selbach M (2012) In vivo quantitative proteome profiling: planning and evaluation of SILAC experiments. Methods Mol Biol 893:175–199
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: 351–364
Acknowledgements
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Soufi, B., Macek, B. (2014). Stable Isotope Labeling by Amino Acids Applied to Bacterial Cell Culture. In: Warscheid, B. (eds) Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). Methods in Molecular Biology, vol 1188. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1142-4_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1142-4_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1141-7
Online ISBN: 978-1-4939-1142-4
eBook Packages: Springer Protocols