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, Drosophila melanogaster 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 D. melanogaster extracts and mouse serum.

Recent Advances in NMR-Based Metabolomics

“…To alleviate this bottleneck, a custom-built 13 C-optimized probe was used to obtain high-quality 13 C NMR spectra. 40 A lower limit of 13 C NMR detection of approximately 40 nmol was obtained, and a combined analysis of 13 C and 1 H data using statistical correlation was shown to improve metabolite identification. Another technique that is often used for small molecule NMR applications is the 13C– 13 C incredible natural abundance double quantum transfer experiment (INADEQUATE), which is a highly useful 2D NMR method for metabolite identification.…”

The future of NMR-based metabolomics

“…Small high-temperature superconducting coils can maximize the signal per sample mass: a 13 C-optimized 1.5-mm high temperature superconducting NMR probe has enabled novel 13 C NMR studies of natural products [47], and this has been followed up with a 1 H- 13 C dual-optimized NMR probe based on double-tuned high temperature superconducting resonators [48]. These probes take advantage of the excellent peak dispersion of 13 C spectra [49], which can be augmented by further 2D 13 C- 13 C experiments, such as INADEQUATE [50]…”

Can NMR solve some significant challenges in metabolomics?

“…STOCSY (statistical total correlation spectroscopy) generates correlation coefficients between every pair of 1D NMR peaks across multiple spectra, and metabolites can then be identified based on peaks showing high correlations [58–62]. A large number of variants of STOCSY have been developed for compound identification, data preprocessing, and metabolic pathway analysis (Fig.…”