Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Protein-based stable isotope probing

Nature Protocols volume 5, pages 1957–1966 (2010)Cite this article

Subjects

Abstract

We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon (13C)- or nitrogen (15N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2–3 d.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overview of the protein-SIP analysis workflow.
Figure 2: Representative peptide mass fingerprint (MS) spectra of benzene 1,2-dioxygenase beta protein subunit from P. putida ML2.

Similar content being viewed by others

References

  1. Crick, F. Central dogma of molecular biology. Nature 227, 561–563 (1970).

    Article  CAS  Google Scholar 

  2. Radajewski, S., Ineson, P., Parekh, N.R. & Murrell, J.C. Stable-isotope probing as a tool in microbial ecology. Nature 403, 646–649 (2000).

    Article  CAS  Google Scholar 

  3. Manefield, M., Whiteley, A.S., Griffiths, R.I. & Bailey, M.J. RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl. Environ. Microbiol. 68, 5367–5373 (2002).

    Article  CAS  Google Scholar 

  4. Boschker, H.T.S. et al. Direct linking of microbial populations to specific biogeochemical processes by C-13-labelling of biomarkers. Nature 392, 801–805 (1998).

    Article  CAS  Google Scholar 

  5. Dumont, M.G. & Murrell, J.C. Stable isotope probing—linking microbial identity to function. Nat. Rev. Microbiol. 3, 499–504 (2005).

    Article  CAS  Google Scholar 

  6. Neufeld, J.D., Dumont, M.G., Vohra, J. & Murrell, J.C. Methodological considerations for the use of stable isotope probing in microbial ecology. Microb. Ecol. 53, 435–442 (2007).

    Article  CAS  Google Scholar 

  7. Evershed, R.P. et al. 13C-Labelling of lipids to investigate microbial communities in the environment. Curr. Opin. Biotechnol. 17, 72–82 (2006).

    Article  CAS  Google Scholar 

  8. Jehmlich, N. et al. Incorporation of carbon and nitrogen atoms into proteins measured by protein-based stable isotope probing (Protein-SIP). Rapid Commun. Mass Spectrom. 22, 2889–2897 (2008).

    Article  CAS  Google Scholar 

  9. Jehmlich, N., Schmidt, F., von Bergen, M., Richnow, H.H. & Vogt, C. Protein-based stable isotope probing (Protein-SIP) reveals active species within anoxic mixed cultures. ISME J. 2, 1122–1133 (2008).

    Article  CAS  Google Scholar 

  10. Bastida, F. et al. Elucidating MTBE degradation in a mixed consortium using a multidisciplinary approach. FEMS Microbiol. Ecol. 73, 370–84 (2010).

    CAS  PubMed  Google Scholar 

  11. Hu, L., Ye, M., Jiang, X., Feng, S. & Zou, H. Advances in hyphenated analytical techniques for shotgun proteome and peptidome analysis—a review. Anal. Chim. Acta. 598, 193–204 (2007).

    Article  CAS  Google Scholar 

  12. Benndorf, D., Balcke, G.U., Harms, H. & von Bergen, M. Functional metaproteome analysis of protein extracts from contaminated soil and groundwater. ISME J. 1, 224–234 (2007).

    Article  CAS  Google Scholar 

  13. Benndorf, D. et al. Improving protein extraction and separation methods for investigating the metaproteome of anaerobic benzene communities within sediments. Biodegradation 20, 737–750 (2009).

    Article  CAS  Google Scholar 

  14. Keller, M. & Hettich, R. Environmental proteomics: a paradigm shift in characterizing microbial activities at the molecular level. Microbiol. Mol. Biol. Rev. 73, 62–70 (2009).

    Article  CAS  Google Scholar 

  15. Purdy, K.J. Nucleic acid recovery from complex environmental samples. Methods Enzymol. 397, 271–292 (2005).

    Article  CAS  Google Scholar 

  16. Jehmlich, N. et al. Comparison of methods for simultaneous identification of bacterial species and determination of metabolic activity by protein-based stable isotope probing (Protein-SIP) experiments. Rapid Commun. Mass Spectrom. 23, 1871–1878 (2009).

    Article  CAS  Google Scholar 

  17. Gustavsson, N. et al. A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry. Proteomics 5, 3563–3570 (2005).

    Article  CAS  Google Scholar 

  18. MacCoss, M.J., Wu, C.C., Matthews, D.E. & Yates, J.R. III . Measurement of the isotope enrichment of stable isotope-labeled proteins using high-resolution mass spectra of peptides. Anal. Chem. 77, 7646–7653 (2005).

    Article  CAS  Google Scholar 

  19. Karty, J.A., Ireland, M.M., Brun, Y.V. & Reilly, J.P. Artifacts and unassigned masses encountered in peptide mass mapping. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 782, 363–383 (2002).

    Article  CAS  Google Scholar 

  20. Fetzer, I. et al. Calculation of partial isotope incorporation into peptides measured by mass spectrometry. BMC Res. Notes 3, 178 (2010).

    Article  Google Scholar 

  21. Jehmlich, N. et al. Decimal place slope, a fast and precise method for quantifying 13C incorporation levels for detecting the metabolic activity of microbial species. Mol. Cell Proteomics 9, 1221–1227 (2010).

    Article  CAS  Google Scholar 

  22. Lueders, T., Manefield, M. & Friedrich, M.W. Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients. Environ. Microbiol. 6, 73–78 (2004).

    Article  CAS  Google Scholar 

  23. Webster, G. et al. A comparison of stable-isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfate-reducing marine sediment enrichment slurries. Environ. Microbiol. 8, 1575–1589 (2006).

    Article  CAS  Google Scholar 

  24. Huang, W.E., Griffiths, R.I., Thompson, I.P., Bailey, M.J. & Whiteley, A.S. Raman microscopic analysis of single microbial cells. Anal. Chem. 76, 4452–4458 (2004).

    Article  CAS  Google Scholar 

  25. Lechene, C.P., Luyten, Y., McMahon, G. & Distel, D.L. Quantitative imaging of nitrogen fixation by individual bacteria within animal cells. Science 317, 1563–1566 (2007).

    Article  CAS  Google Scholar 

  26. Musat, N. et al. A single-cell view on the ecophysiology of anaerobic phototrophic bacteria. Proc. Natl. Acad. Sci. USA 105, 17861–17866 (2008).

    Article  CAS  Google Scholar 

  27. Macias, M.T. Use of radionuclides in cancer research and treatment. Clin. Transl. Oncol. 11, 143–153 (2009).

    Article  CAS  Google Scholar 

  28. Nikolausz, M. et al. Novel approach using substrate-mediated radiolabelling of RNA to link metabolic function with the structure of microbial communities. FEMS Microbiol. Lett. 274, 154–161 (2007).

    Article  CAS  Google Scholar 

  29. Tabb, D.L. et al. Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry. J. Proteome Res. 9, 761–776 (2009).

    Article  Google Scholar 

  30. Craig, R. & Beavis, R.C. TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20, 1466–1467 (2004).

    Article  CAS  Google Scholar 

  31. Snijders, A.P.L., de Vos, M.G.J., de Koning, B. & Wright, P.C. A fast method for quantitative proteomics based on a combination between two-dimensional electrophoresis and N-15-metabolic labelling. Electrophoresis 26, 3191–3199 (2005).

    Article  CAS  Google Scholar 

  32. Senko, M.W., Beu, S.C. & McLafferty, F.W. Determination of monoisotopic masses and ion populations for large biomolecules. Am. Soc. Mass Spectrom. 6, 229–233 (1995).

    Article  CAS  Google Scholar 

  33. Widdel, F. & Rabus, R. Anaerobic biodegradation of satured and aromatic hydrocarbons. Curr. Opin. Biotechnol. 12, 259–276 (2001).

    Article  CAS  Google Scholar 

  34. Rabus, R. & Widdel, F. Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria. Arch. Microbiol. 163, 96–103 (1995).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by funding from the Helmholtz Association. M.T. was funded by a grant from the Priority Program SPP1319 from the German Research Association. F.B. acknowledges support from Marie Curie Mobility Actions of the European Commission: Host Fellowship for the Transfer of Knowledge (ToK) ISOTONIC project (MTKD-CT-2006-042758).

Author information

Authors and Affiliations

  1. Department of Proteomics, Helmholtz-Centre for Environmental Research – UFZ, Leipzig, Germany

    Nico Jehmlich, Martin Taubert, Jana Seifert & Martin von Bergen

  2. Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany

    Nico Jehmlich & Frank Schmidt

  3. Department of Isotope Biogeochemistry, Helmholtz-Centre for Environmental Research – UFZ, Leipzig, Germany

    Felipe Bastida, Hans-Hermann Richnow & Carsten Vogt

  4. Department of Soil and Water Conservation and Waste Management, CEBAS-CSIC, Campus Universitario de Espinardo, Murcia, Spain

    Felipe Bastida

  5. Department of Metabolomics, Helmholtz-Centre for Environmental Research – UFZ, Leipzig, Germany

    Martin von Bergen

Authors
  1. Nico Jehmlich
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frank Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin Taubert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jana Seifert
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Felipe Bastida
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Martin von Bergen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hans-Hermann Richnow
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Carsten Vogt
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.J. participated in experimental analysis, data analysis and writing; F.S. helped with data analysis and writing; M.T. took part in experimental analysis and writing; J.S. was involved in data analysis and writing; F.B. conducted experimental analysis and data analysis; M.v.B. and H.-H.R. were involved in conceptualizing and designing experiments and writing; C.V. participated in experimental design, experimental analysis, data analysis and writing.

Corresponding author

Correspondence to Martin Taubert.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jehmlich, N., Schmidt, F., Taubert, M. et al. Protein-based stable isotope probing. Nat Protoc 5, 1957–1966 (2010). https://doi.org/10.1038/nprot.2010.166

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.166

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research