Quantitative Analysis of Metabolic Mixtures by Two-Dimensional 13C Constant-Time TOCSY NMR Spectroscopy

An increasing number of organisms can be fully C-13-labeled, which has the advantage that their metabolomes can be studied by high resolution two-dimensional (2D) NMR C-13-C-13 constant time (CT) total correlation spectroscopy (TOCSY) experiments. Individual metabolites can be identified via database searching or, in the case of novel compounds, through the reconstruction of their backbone carbon topology. Determination of quantitative metabolite concentrations is another key task. Because strong peak overlaps in one-dimensional (1D) NMR spectra prevent straightforward quantification through ID peak integrals, we demonstrate here the direct use of C-13-C-13 CT-TOCSY spectra for metabolite quantification. This is accomplished through the quantum mechanical treatment of the TOCSY magnetization transfer at short and long mixing times or by the use of analytical approximations, which are solely based on the knowledge of the carbon-backbone topologies. The methods are demonstrated for carbohydrate and amino acid mixtures.