Kinetic flux profiling for quantitation of cellular metabolic fluxes

241 Carl Icahn Laboratory, Department of Chemistry and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, USA.
Nature Protocol (Impact Factor: 9.67). 02/2008; 3(8):1328-40. DOI: 10.1038/nprot.2008.131
Source: PubMed


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)<or=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 approximately 15 metabolites can be obtained over 3 d of work. Applications to adherent mammalian cells are also discussed.

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    • "Note also that the choice of interconnection signals between subsystems is independent of the choice of the controlled output of the process: one can define flux variables as interconnection signals and a concentration variable as controlled output. This is important, because the measurement of molecular fluxes, compared to concentrations, is still a difficult task from the technological point of view; in metabolic engineering, for example, it is achieved through the incorporation of stable isotopes from nutrient into downstream metabolites [12]. "
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    • "More explicitly, the rate of labeling of a metabolite pool is dependent upon the following: (1) the extent of labeling of the precursor substrate molecule(s) as label propagates through the system; (2) the size of the metabolite pool (a large pool will take longer to become fully labeled than a small one); and (3) the rate of conversion of precursor substrate(s) into the metabolite (the desired flux parameter). In the simple case of an irreversible monomolecular reaction, it is possible to estimate the flux by fitting the reactant labeling profiles to the solution of a single differential equation that describes these relationships, in an approach known as kinetic flux profiling (Yuan et al., 2006, 2008). However, in most cases, the complexity of the network increases the number of parameters that must be considered and the equations are no longer analytically solvable. "
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