¹³C NMR Metabolomics: Applications at Natural Abundance

Abstract

¹³C NMR has many advantages for a metabolomics study, including a large spectral dispersion, narrow singlets at natural abundance, and a direct measure of the backbone structures of metabolites. However, it has not had widespread use because of its relatively low sensitivity compounded by low natural abundance. Here we demonstrate the utility of high-quality ¹³C NMR spectra obtained using a custom ¹³C-optimized probe on metabolomic mixtures. A workflow was developed to use statistical correlations between replicate 1D ¹³C and ¹H spectra, leading to composite spin systems that can be used to search publicly available databases for compound identification. This was developed using synthetic mixtures and then applied to two biological samples, <i>Drosophila melanogaster</i> extracts and mouse serum. Using the synthetic mixtures we were able to obtain useful ¹³C–¹³C statistical correlations from metabolites with as little as 60 nmol of material. The lower limit of ¹³C NMR detection under our experimental conditions is approximately 40 nmol, slightly lower than the requirement for statistical analysis. The ¹³C and ¹H data together led to 15 matches in the database compared to just 7 using ¹H alone, and the ¹³C correlated peak lists had far fewer false positives than the ¹H generated lists. In addition, the ¹³C 1D data provided improved metabolite identification and separation of biologically distinct groups using multivariate statistical analysis in the <i>D. melanogaster</i> extracts and mouse serum