Kinetic flux profiling for quantitation of cellular metabolic fluxes

Jie Yuan, Bryson D. Bennett, Joshua D. Rabinowitz

Research output: Contribution to journalArticle

172 Scopus citations

Abstract

This protocol enables quantitation of metabolic fluxes in cultured cells. Measurements are based on the kinetics of cellular incorporation of stable isotope from nutrient into downstream metabolites. At multiple time points, after cells are rapidly switched from unlabeled to isotope-labeled nutrient, metabolism is quenched, metabolites are extracted and the extract is analyzed by chromatography-mass spectrometry. Resulting plots of unlabeled compound versus time follow variants of exponential decay, with the flux equal to the decay rate multiplied by the intracellular metabolite concentration. Because labeling is typically fast (t1/2 ≤5 min for central metabolites in Escherichia coli), variations on this approach can effectively probe dynamically changing metabolic fluxes. This protocol is exemplified using E. coli and nitrogen labeling, for which quantitative flux data for ∼15 metabolites can be obtained over 3 d of work. Applications to adherent mammalian cells are also discussed.

Original languageEnglish (US)
Pages (from-to)1328-1340
Number of pages13
JournalNature Protocols
Volume3
Issue number8
DOIs
StatePublished - 2008

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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