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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Sauer, U. (2004) High-throughput phenomics: experimental methods for map** fluxomes. Curr. Opin. Biotechnol. 15, 58–63.
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.
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.
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.
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.
Fuhrer, T., Fischer, E., and Sauer, U. (2005) Experimental identification and quantification of glucose metabolism in seven bacterial species. J. Bacteriol. 187, 1581–1590.
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.
Zamboni, N., Fischer, E., and Sauer, U. (2005) Fiat Flux: a software for metabolic flux analysis from 13C-glucose experiments. BMC Bioinformatics 6, 209.
Dauner, M. and Sauer, U. (2000) GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing. Biotechnol. Prog. 16, 642–649.
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.
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.
Rosman, K. J. R. and Taylor, P. D. P. (1998) Isotopic compositions of the elements 1997. Pure Appl. Chem. 70, 217–235.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Nanchen, A., Fuhrer, T., Sauer, U. (2007). Determination of Metabolic Flux Ratios From 13C-Experiments and Gas Chromatography-Mass Spectrometry Data. In: Weckwerth, W. (eds) Metabolomics. Methods in Molecular Biology™, vol 358. Humana Press. https://doi.org/10.1007/978-1-59745-244-1_11
Download citation
DOI: https://doi.org/10.1007/978-1-59745-244-1_11
Publisher Name: Humana Press
Print ISBN: 978-1-58829-561-3
Online ISBN: 978-1-59745-244-1
eBook Packages: Springer Protocols