Research for the Environment

Protein stable-isotope probing (Protein-SIP) for functional metaproteomics

Project description

The Protein-SIP technique (SIP = stable isotope probing) was established in cooperation with the Department of Isotope Biogeochemistry to characterise microbial communities. This method allows a concomitant link between genera identification and metabolic activity. Thus, the phylogenic and functional investigations of microbial communities are possible.

1 Differential 13C incorporation by assimilation

Assimilation of heavy stable isotopes into the biomass of various species depends on the turnover and the metabolic activity of the species. The incorporation of stable 13C isotopes from a substrate can be used to pinpoint the metabolically active species within a consortium. The different incorporation levels are indicated by the intensity of blue staining.

2 Reference curves enable quantification

After cell harvesting and protein extraction, samples were tryptically digested and analyzed by mass spectrometry. The isotopologues shifted to a higher mass range due to their incorporation of heavily labeled carbon into the proteins. A higher level of incorporation prooves a higher metabilisation and thereby a primary role in the usage of the labelled subtrate within the food web.
The incorporation of heavy stable isotopes into peptides/proteins can be estimated using the half decimal place rule.

3 Interdependency in food web analysed by carbon flux

Peptides can be used to obtain phylogenetic information, for structural analysis of the consortium, and for physiological information about the actual state of the microbial cells.
Based on this information, C-fluxes and food web structures can be elucidated and may further help to reconstruct the interactions within microbial communities.

Prinicple of Protein-SIP

Fig.1 Principle of Protein-SIP

Involved researchers


  • Carsten Vogt, Petra Bombach, Hans-Hermann Richnow; Dep. Isotopenbiogeochemie
  • Brandon E. Morris, Josef M. Suflita, University of Oklahoma
  • Michael Siegert, Martin Krüger, BGR Hannover
  • Sara Kleindienst, Katrin Knittel, MPI für Marine Mikrobiologie Bremen
  • Sascha Krause, Paul Bodelier; Netherlands Institute of Ecology
  • Michael Pester, University Vienna


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

Jehmlich N, Schmidt F, Hartwich M, von Bergen M, Richnow HH, Vogt C (2008) Incorporation of carbon and nitrogen atmos into proteins measured by protein-based stable isotope probing (Protein-SIP).
Rapid Commun Mass Spectrom. 22: 2889-2897.

Jehmlich N, Schmidt F, Taubert M, Seifert J, von Bergen M, Richnow HH, Vogt C (2009) 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.

Jehmlich N, Fetzer I, Seifert J, Mattow J, Vogt C, Harms H, Thiede B, Richnow HH, von Bergen M, Schmidt F (2010) 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.

Fetzer I, Jehmlich N, Vogt C, Richnow HH, Seifert J, Harms H, von Bergen M, Schmidt F (2010) Calculation of partial isotope incorporation into peptides measured by mass spectrometry.
BMC Res Notes. Jun 24;3(1):178.

Bastida F, Rosell M, Franchini AG, Seifert J, Finsterbusch S, Jehmlich J, Jechalke S, von Bergen M, Richnow HH (2010) Elucidating MTBE degradation in a mixed consortium using a multidisciplinary approach.
FEMS Microbiol Ecol. 73(2):370-84.

Jehmlich N, Schmidt F, Taubert M, Seifert J, Bastida F, von Bergen M, Richnow HH, Vogt C (2010) Protein-stable isotope probing (Protein-SIP).
Nature Protocols. 5 (12), 1957-1966.

Jehmlich N, Seifert J, Taubert M, Schmidt F, Vogt C, Richnow HH, von Bergen M (2011) Protein Stable Isotope Probing.
In: Murrell JC, Whiteley AS (eds) Stable isotope probing and related technologies. ASM Press, Washington

Taubert, M, Baumann, S, von Bergen, M, Seifert, J (2011) Exploring the limits of robust detection of incorporation of 13C by mass spectrometry in protein-based stable isotope probing (protein-SIP).
Anal. Bioanal. Chem. 401 (6), 1975-1982.

Bastida, F, Jechalke, S, Bombach, P, Franchini, AG, Seifert, J, von Bergen, M, Vogt, C, Richnow, HH (2011) Assimilation of benzene carbon through multiple trophic levels traced by different stable isotope probing methodologies.
FEMS Microbiol Ecol. 77 (2), 357-369.

Taubert, M, Jehmlich, N, Schmidt, F, Vogt, C, Richnow, HH, von Bergen, M, Seifert, J (2011) Time resolved protein-based stable isotope probing (Protein-SIP) analysis allows quantification of induced proteins in substrate shift experiments.
Proteomics. 11 (11), 2265–2274.

Jehmlich, N, Kopinke, FD, Lenhard, S, Herbst, FA, Seifert, J, Lissner, U, Völker, U, Schmidt, F, von Bergen, M. (2012) Sulphur-36S stable isotope labeling of amino acids for quantification (SULAQ).
Proteomics. 12 (1), 37-42.

Morris, BEL, Herbst, FA, Bastida, F, Seifert, J, von Bergen, M, Richnow, HH, Suflita, J (2012) Microbial interactions during residual oil and n-fatty acid metabolism by a methanogenic consortium.
Env. Microbiol. Rep. 4(3), 297-306

Kermer, R, Hedrich, S, Taubert, M, Baumann, S, Schlömann, M, Johnson, DB, von Bergen, M, Seifert, J (2012) Elucidation of carbon transfer in a mixed culture of Acidiphilium cryptum and Acidithiobacillus ferrooxidans using protein-based stable isotope probing.
JIOMICS. 2(1): 37-45. doi: 10.5584/jiomics.v2012i2012.85

Bozinovski, D, Herrmann, S, Richnow, HH, von Bergen, M, Seifert, J, Vogt, C (2012) Functional analysis of an anaerobic m-xylene degrading enrichment culture using protein-based stable isotope probing.
FEMS Microbiol. Ecol. 81(1):134-144

Seifert, J, Taubert, M, Jehmlich, N, Schmidt, F, Völker, U, Vogt, C, Richnow, HH, von Bergen, M (2012) Protein-based stable isotope probing in functional metaproteomics.
Mass Spectrom. Rev. 31 (6), 683-697

Marco-Urrea, E, Seifert, J, von Bergen, M, Lorenz, A (2012) Application of stable isotope peptide mass spectrometry to decipher amino acid metabolism in Dehalococcoides strain CBDB1.
J. Bact. 194 (16), 4169-4177

Taubert, M, Vogt, C, Wubet, T, Kleinsteuber, S, Tarkka, MT, Harms, H, Buscot, F, Richnow, HH, von Bergen, M, Seifert, J (2012) Protein-SIP enables time-resolved analysis of the carbon flux in a sulfate-reducing, benzene-degrading microbial consortium.
ISME J. 6 (12), 2291 - 2301