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

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...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2016-03, Vol.88 (5), p.2825-2831
Main Authors: Reardon, P. N, Marean-Reardon, C. L, Bukovec, M. A, Coggins, B. E, Isern, N. G
Format: Article
Language:English
Subjects:
Enrichment
Environmental Molecular Sciences Laboratory
Experiments
Flux
Fluxes
INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
Metabolism
Networks
NMR
Nuclear magnetic resonance
Sampling
Spectrum analysis
Symbols
Three dimensional
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Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.5b04535