Fast Spatially Encoded 3D NMR Strategies for C-13-Based Metabolic Flux Analysis

The measurement of site-specific C-13 enrichments in complex mixtures of C-13-labeled metabolites is a powerful tool for metabolic flux analysis. One of the main methods to measure such enrichments is homonuclear H-1 2D NMR However, the major limitation of this technique is the acquisition time, whi...

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Published inAnalytical chemistry (Washington) Vol. 85; no. 20; pp. 9751 - 9757
Main Authors Boisseau, Renaud, Charrier, Benoit, Massou, Stéphane, Portais, Jean-Charles, Akoka, Serge, Giraudeau, Patrick
Format Journal Article
LanguageEnglish
Published American Chemical Society 15.10.2013
Subjects
Life Sciences
ENRICHMENTS
PHYSIOLOGY
INTERMEDIARY METABOLISM
C-13 ISOTOPOMER ANALYSIS
NUCLEAR-MAGNETIC-RESONANCE
ULTRAFAST 2D NMR
SENSITIVITY LOSSES
J-RESOLVED SPECTROSCOPY
SPECTRA
SINGLE-SCAN
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Summary:The measurement of site-specific C-13 enrichments in complex mixtures of C-13-labeled metabolites is a powerful tool for metabolic flux analysis. One of the main methods to measure such enrichments is homonuclear H-1 2D NMR However, the major limitation of this technique is the acquisition time, which can amount to a few hours. This drawback was recently overcome by the design of fast COSY experiments for measuring specific C-13-enrichments, based on single-scan 2D NMR However, these experiments are still limited by overlaps because of H-1-C-13 splittings, thus limiting the metabolic information accessible for complex biological mixtures. To circumvent this limitation, we propose to tilt the H-1-C-13 coupling into a third dimension via fast-hybrid 3D NMR methods combining the speed of ultrafast 2D NMR with the high resolution of conventional methods. Two strategies are described that allow the acquisition of a complete 3D J-resolved-COSY spectrum in 12 min (for concentrations as low as 10 mM). The analytical potentialities of both methods are evaluated on a series of C-13-enriched glucose samples and on a biomass hydrolyzate obtained from Escherichia coli cells. Once optimized, the two complementary experiments lead to a trueness and a precision of a few percent and an excellent linearity. The advantages and drawbacks of these approaches are discussed and their potentialities are highlighted.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac402155w