Metabolic Labeling of Microorganisms with Stable Heavy Nitrogen Isotopes (15N)

  • Andreas OttoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1841)


Quantitative Proteomics based on stable isotope labeling by 15N is a versatile method in determination of changes in microbial protein expression profiles. Once set up, the workflow relies on defined chemical or commercially available complex media leading to universal heavy labeling by the stable isotope 15N. This chapter provides a detailed protocol for labeling of different bacteria and the strategy for evaluating the incorporation rate of the heavy stable isotopes in the sample is discussed.

Key words

Microorganisms Gel-free proteomics Quantitative proteomics 15



This work was supported by the German Research Foundation Grant SFB/TR34.


  1. 1.
    Sabido E, Selevsek N, Aebersold R (2012) Mass spectrometry-based proteomics for systems biology. Curr Opin Biotechnol 23(4):591–597CrossRefPubMedGoogle Scholar
  2. 2.
    Cravatt BF, Simon GM, Yates JR 3rd (2007) The biological impact of mass-spectrometry-based proteomics. Nature 450(7172):991–1000CrossRefPubMedGoogle Scholar
  3. 3.
    Bantscheff M, Lemeer S, Savitski MM, Küster B (2012) Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present. Anal Bioanal Chem 404(4):939–965CrossRefPubMedGoogle Scholar
  4. 4.
    Bantscheff M, Schirle M, Sweetman G, Rick J, Küster B (2007) Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem 389(4):1017–1031CrossRefPubMedGoogle Scholar
  5. 5.
    Gouw JW, Krijgsveld J, Heck AJ (2010) Quantitative proteomics by metabolic labeling of model organisms. Mol Cell Proteomics 9(1):11–24CrossRefPubMedGoogle Scholar
  6. 6.
    Otto A, Becher D, Schmidt F (2014) Quantitative proteomics in the field of microbiology. Proteomics 14(4–5):547–565CrossRefPubMedGoogle Scholar
  7. 7.
    Otto A, Bernhardt J, Meyer H, Schaffer M, Herbst FA, Siebourg J, Mäder U, Lalk M, Hecker M, Becher D (2010) Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis. Nat Commun 1:137CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Becher D, Hempel K, Sievers S, Zühlke D, Pané-Farré J, Otto A, Fuchs S, Albrecht D, Bernhardt J, Engelmann S, Völker U, van Dijl JM, Hecker M (2009) A proteomic view of an important human pathogen–towards the quantification of the entire Staphylococcus aureus proteome. PLoS One 4(12):e8176CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Dreisbach A, Otto A, Becher D, Hammer E, Teumer A, Gouw JW, Hecker M, Völker U (2008) Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques. Proteomics 8(10):2062–2076CrossRefPubMedGoogle Scholar
  10. 10.
    Eng JK, Jahan TA, Hoopmann MR (2013) Comet: an open-source MS/MS sequence database search tool. Proteomics 13(1):22–24CrossRefPubMedGoogle Scholar
  11. 11.
    Price CE, Otto A, Fusetti F, Becher D, Hecker M, Driessen AJ (2010) Differential effect of YidC depletion on the membrane proteome of Escherichia coli under aerobic and anaerobic growth conditions. Proteomics 10(18):3235–3247CrossRefPubMedGoogle Scholar
  12. 12.
    Filiou MD, Varadarajulu J, Teplytska L, Reckow S, Maccarrone G, Turck CW (2012) The 15N isotope effect in Escherichia coli: a neutron can make the difference. Proteomics 12(21):3121–3128CrossRefPubMedGoogle Scholar
  13. 13.
    Frank E, Kessler MS, Filiou MD, Zhang Y, Maccarrone G, Reckow S, Bunck M, Heumann H, Turck CW, Landgraf R, Hambsch B (2009) Stable isotope metabolic labeling with a novel N-enriched bacteria diet for improved proteomic analyses of mouse models for psychopathologies. PLoS One 4(11):e7821CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    MacCoss MJ, Wu CC, Liu H, Sadygov R, Yates JR 3rd (2003) A correlation algorithm for the automated quantitative analysis of shotgun proteomics data. Anal Chem 75(24):6912–6921CrossRefPubMedGoogle Scholar
  15. 15.
    Geiger T, Wisniewski JR, Cox J, Zanivan S, Kruger M, Ishihama Y, Mann M (2011) Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics. Nat Protoc 6(2):147–157CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Microbial Proteomics, Institute for MicrobiologyUniversity GreifswaldGreifswaldGermany

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