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

Reardon, P. N., Marean-Reardon, C. L., Bukovec, M. A., Coggins, B. E., Isern, N. G.
Analytical chemistry 2016 v.88 no.5 pp. 2825-2831
biomass, carbon, hydrolysates, isotope labeling, metabolic flux analysis, metabolites, nuclear magnetic resonance spectroscopy, stable isotopes
¹³C-Metabolic Flux Analysis (¹³C-MFA) is rapidly being recognized as the authoritative method for determining fluxes through metabolic networks. Site-specific ¹³C enrichment information obtained using NMR spectroscopy is a valuable input for ¹³C-MFA experiments. Chemical shift overlaps in the 1D or 2D NMR experiments typically used for ¹³C-MFA frequently hinder assignment and quantitation of site-specific ¹³C enrichment. Here we propose the use of a 3D TOCSY-HSQC experiment for ¹³C-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 ¹³C-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 ¹³C chemical shift obtained in the experiment. In addition, the experiment reports ¹³C-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.