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

Author:
Reardon, P. N., Marean-Reardon, C. L., Bukovec, M. A., Coggins, B. E., Isern, N. G.
Source:
Analytical chemistry 2016 v.88 no.5 pp. 2825-2831
ISSN:
1520-6882
Subject:
biomass, carbon, hydrolysates, isotope labeling, metabolic flux analysis, metabolites, nuclear magnetic resonance spectroscopy, stable isotopes
Abstract:
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.
Agid:
5304522