Journal
NATURE PROTOCOLS
Volume 3, Issue 8, Pages 1328-1340Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2008.131
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Funding
- Beckman Foundation
- NSF DDDAS [CNS-0540181]
- American Heart Association [0635188N]
- NSF CAREER Award [MCB-0643859]
- NIH [AI078063, GM071508]
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R01AI078063] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [P50GM071508] Funding Source: NIH RePORTER
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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 (t(1/2) <= 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 similar to 15 metabolites can be obtained over 3 d of work. Applications to adherent mammalian cells are also discussed.
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