Metabolomics pp 177-197

Part of the Methods in Molecular Biology™ book series (MIMB, volume 358)

Determination of Metabolic Flux Ratios From 13C-Experiments and Gas Chromatography-Mass Spectrometry Data

Protocol and Principles
  • Annik Nanchen
  • Tobias Fuhrer
  • Uwe Sauer

Abstract

Network topology is a necessary fundament to understand function and properties of microbial reaction networks. A valuable method for experimental elucidation of metabolic network topology is metabolic flux ratio analysis, which quantifies the relative contribution of two or more converging pathways to a given metabolite. It is based on 13C-labeling experiments, gas chromatography-mass spectrometry analysis, and probabilistic equations that relate mass distributions in proteinogenic amino acids to pathway activity. Here, we describe the protocol for sample generation and illustrate the principles underlying the calculation of metabolic flux ratios with three examples. These principles are also implemented in the publicly available software FiatFlux, which directly calculates flux ratios from the mass spectra of amino acids.

References

  1. 1.
    Sauer, U. (2004) High-throughput phenomics: experimental methods for mapping fluxomes. Curr. Opin. Biotechnol. 15, 58–63.PubMedCrossRefGoogle Scholar
  2. 2.
    Szyperski, T., Glaser, R. W., Hochuli, M., et al. (1999) Bioreaction network topology and metabolic flux ratio analysis by biosynthetic fractional 13C labeling and two-dimensional NMR spectroscopy. Metab. Eng. 1, 189–197.PubMedCrossRefGoogle Scholar
  3. 3.
    Szyperski, T. (1995) Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism. Eur. J. Biochem. 232, 433–448.PubMedCrossRefGoogle Scholar
  4. 4.
    Fischer, E. and Sauer, U. (2003) Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. Eur. J. Biochem. 270, 880–891.PubMedCrossRefGoogle Scholar
  5. 5.
    Sauer, U., Lasko, D. R., Fiaux, J., et al. (1999) Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism. J. Bacteriol. 181, 6679–6688.PubMedGoogle Scholar
  6. 6.
    Fuhrer, T., Fischer, E., and Sauer, U. (2005) Experimental identification and quantification of glucose metabolism in seven bacterial species. J. Bacteriol. 187, 1581–1590.PubMedCrossRefGoogle Scholar
  7. 7.
    Fischer, E., Zamboni, N., and Sauer, U. (2004) High-throughput metabolic flux analysis based on gas chromatography-mass spectrometry derived 13C constraints. Anal. Biochem. 325, 308–316.PubMedCrossRefGoogle Scholar
  8. 8.
    Zamboni, N., Fischer, E., and Sauer, U. (2005) Fiat Flux: a software for metabolic flux analysis from 13C-glucose experiments. BMC Bioinformatics 6, 209.PubMedCrossRefGoogle Scholar
  9. 9.
    Dauner, M. and Sauer, U. (2000) GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing. Biotechnol. Prog. 16, 642–649.PubMedCrossRefGoogle Scholar
  10. 10.
    Van Winden, W. A., Wittmann, C., Heinzle, E., and Heijnen, J. J. (2002) Correcting mass isotopomer distributions for naturally occurring isotopes. Biotechnol. Bioeng. 80, 477–479.PubMedCrossRefGoogle Scholar
  11. 11.
    Hellerstein, M. K. and Neese, R. A. (1999) Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations. Am. J. Physiol. 276, E1146–E1170.PubMedGoogle Scholar
  12. 12.
    Rosman, K. J. R. and Taylor, P. D. P. (1998) Isotopic compositions of the elements 1997. Pure Appl. Chem. 70, 217–235.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Annik Nanchen
    • 1
  • Tobias Fuhrer
    • 1
  • Uwe Sauer
    • 1
  1. 1.Institute for Molecular Systems BiologyETH ZürichZürichSwitzerland

Personalised recommendations