Quantitative Analysis of Metabolic Mixtures by 2D 13C-Constant-Time TOCSY NMR Spectroscopy

摘要

An increasing number of organisms can be fully ¹³C-labeled, which has the advantage that their metabolomes can be studied by high-resolution 2D NMR ¹³C–¹³C constant-time (CT) 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 significant peak overlaps in 1D NMR spectra prevents straightforward quantification through 1D peak integrals, we demonstrate here the direct use of ¹³C–¹³C 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.