title: Fast spatially encoded 3D NMR strategies for (13)C-based metabolic flux analysis.
abstract: The measurement of site-specific (13)C enrichments in complex mixtures of (13)C-labeled metabolites is a powerful tool for metabolic flux analysis. One of the main methods to measure such enrichments is homonuclear (1)H 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 (13)C-enrichments, based on single-scan 2D NMR. However, these experiments are still limited by overlaps because of(1)H-(13)C splittings, thus limiting the metabolic information accessible for complex biological mixtures. To circumvent this limitation, we propose to tilt the (1)H-(13)C 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 (13)C-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.
authors.affiliation: Université de Nantes , CNRS, CEISAM UMR 6230, B.P. 92208, 2 rue de la Houssinière, 44322 Nantes Cedex 03, France.
authors.forename: Renaud
authors.initials: R
authors.lastname: Boisseau
authors.forename: Benoît
authors.initials: B
authors.lastname: Charrier
authors.forename: Stéphane
authors.initials: S
authors.lastname: Massou
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Portais
authors.forename: Serge
authors.initials: S
authors.lastname: Akoka
authors.forename: Patrick
authors.initials: P
authors.lastname: Giraudeau
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry; D004926:Escherichia coli / Q000166:cytology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D064688:Metabolic Flux Analysis / Q000379:methods*; D013997:Time Factors
chemical_list: D002247:Carbon Isotopes