Abstract: This disclosure provides for new methods for quantification of metabolite concentrations in metabolomics studies, which addresses the difficulties in quantification through 1D peak integrals due to significant peak overlaps in metabolomics samples. For samples from uniformly 13C-labeled organisms the 2D NMR 13C-13C constant-time (CT) TOCSY experiment provides high-resolution information about individual metabolites that allows their identification via database searching or, in the case of novel compounds, through the reconstruction of their backbone-carbon topology. This disclosure further demonstrates using CT-TOCSY spectra for quantification purposes.

Abstract: This disclosure provides for new methods for quantification of metabolite concentrations in metabolomics studies, which addresses the difficulties in quantification through 1D peak integrals due to significant peak overlaps in metabolomics samples. For samples from uniformly 13C-labeled organisms the 2D NMR 13C-13C constant-time (CT) TOCSY experiment provides high-resolution information about individual metabolites that allows their identification via database searching or, in the case of novel compounds, through the reconstruction of their backbone-carbon topology. This disclosure further demonstrates using CT-TOCSY spectra for quantification purposes.

Abstract: This disclosure provides new multidimensional-NMR approaches that are useful in the analysis of mixtures with high complexity at natural 13C abundance, including ones encountered in metabolomics. Common to all three approaches is the concept of the extraction of 1D consensus spectral traces or 2D consensus planes followed by clustering, which significantly improves the capability to identify mixture components affected by strong spectral overlap. The methods are demonstrated for covariance 1H-1H TOCSY and 13C-1H HSQC-TOCSY spectra and triple-rank correlation spectra constructed from pairs of 13C-1H HSQC and 13C-1H HSQC-TOCSY spectra. All methods are demonstrated for a metabolite model mixture and then applied to an extract from E. coli cell lysate. This disclosure also provides a homonuclear 13C 2D NMR approach, namely CT-TOCSY, which is applied to a non-fractionated uniformly 13C-enriched lysate of E. coli cells to determine de novo the carbon backbone topologies that constitute their “topolome”.