13C-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.