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(13)C-Metabolic Flux Analysis ((13)C-MFA) is rapidly being recognized as the authoritative method for determining fluxes through metabolic networks. Site-specific (13)C enrichment information obtained using NMR spectroscopy is a valuable input for (13)C-MFA experiments. Chemical shift overlaps in the 1D or 2D NMR experiments typically used for (13)C-MFA frequently hinder assignment and quantitation of site-specific (13)C enrichment. Here we propose the use of a 3D TOCSY-HSQC experiment for (13)C-MFA. We employ Non-Uniform Sampling (NUS) to reduce the acquisition time of the experiment to a few hours, making it practical for use in (13)C-MFA experiments. Our data show that the NUS experiment is linear and quantitative. Identification of metabolites in complex mixtures, such as a biomass hydrolysate, is simplified by virtue of the (13)C chemical shift obtained in the experiment. In addition, the experiment reports (13)C-labeling information that reveals the position specific labeling of subsets of isotopomers. The information provided by this technique will enable more accurate estimation of metabolic fluxes in large metabolic networks.

3D TOCSY-HSQC NMR for Metabolic Flux Analysis Using Non-Uniform Sampling