Comparison of methods for simultaneous identification of bacterial species and determination of metabolic activity by protein-based stable isotope probing (Protein-SIP) experiments
Article first published online: 15 MAY 2009
Copyright © 2009 John Wiley & Sons, Ltd.
Rapid Communications in Mass Spectrometry
Volume 23, Issue 12, pages 1871–1878, 30 June 2009
How to Cite
Jehmlich, N., Schmidt, F., Taubert, M., Seifert, J., von Bergen, M., Richnow, H.-H. and 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. doi: 10.1002/rcm.4084
- Issue published online: 15 MAY 2009
- Article first published online: 15 MAY 2009
- Manuscript Revised: 9 APR 2009
- Manuscript Accepted: 9 APR 2009
- Manuscript Received: 6 NOV 2008
- European Commission, Marie Curie Contract. Grant Number: MTKD-CT 2006-042758
- DFG Priority Programme 1319
We developed a concept for analysing carbon and nitrogen fluxes in microbial communities by employing protein-based stable isotope probing (Protein-SIP) in metabolic labelling experiments with stable isotope labelled substrates. For identification of microbial species intact protein profiling (IPP) can be used, whereas the assessment of their metabolic activity is achieved by shotgun mass mapping (SMM). Microbial cultures were grown on substrates containing 13C or 15N. For identification of species we tested both the IPP and the SMM approaches. Mass spectra (MALDI-MS) were taken from mixtures of either intact proteins or peptides from tryptic digestion for generating species-specific peak patterns. In the case of SMM, the fragmentation of peptides was additionally used to obtain sequence information for species identification. Mass spectra of peptide sequences allow calculation of the amount of 13C or 15N incorporation within peptides for determining metabolic activity of the specific species. The comparison of IPP and SMM revealed a higher robustness of species identification by SMM. In addition, the assessment of incorporation levels of 13C and 15N into peptides by SMM revealed a lower uncertainty (0.5–0.8 atom %) compared to IPP (6.4–8.9 atom %). The determination of metabolic activity and function of individual species by Protein-SIP can help to analyse carbon and nitrogen fluxes within microbial communities. Copyright © 2009 John Wiley & Sons, Ltd.