13 C-based metabolic flux analysis

Stable isotope, and in particular 13 C-based flux analysis, is the exclusive approach to experimentally quantify the integrated responses of metabolic networks. Here we describe a protocol that is based on growing microbes on 13 C-labeled glucose and subsequent gas chromatography mass spectrometric detection of 13 C-patterns in protein-bound amino acids. Relying on publicly available software packages, we then describe two complementary mathematical approaches to estimate either local ratios of converging fluxes or absolute fluxes through different pathways. As amino acids in cell protein are abundant and stable, this protocol requires a minimum of equipment and analytical expertise. Most other flux methods are variants of the principles presented here. A true alternative is the analytically more demanding dynamic flux analysis that relies on 13 C-pattern in free intracellular metabolites. The presented protocols take 5–10 d, have been used extensively in the past decade and are exemplified here for the central metabolism of Escherichia coli.

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Acknowledgements

We thank Katharina Nöh and Wolfgang Wiechert for support with 13CFLUX and comments on the respective protocol parts presented in this protocol, as well as Roelco J. Kleijn, Dominik Heer, Julian Schnidder, and Daniel Heine for constructive comments on the script.

Author information

Authors and Affiliations

  1. Institute of Molecular Systems Biology, ETH Zurich, Zurich, 8093, Switzerland Nicola Zamboni, Sarah-Maria Fendt, Martin Rühl & Uwe Sauer
  2. PhD Program Systems Biology of Complex Diseases, ETH Zurich, Zurich, Switzerland Sarah-Maria Fendt
  3. PhD Program Molecular Life Sciences, ETH Zurich, Zurich, Switzerland Martin Rühl
  1. Nicola Zamboni