Phosphorus NMR and Its Application to Metabolomics

Stable isotopes are routinely employed by NMR metabolomics to highlight specific metabolic processes and to monitor pathway flux. C-13-carbon and N-15-nitrogen labeled nutrients are convenient sources of isotope tracers and are commonly added as supplements to a variety of biological systems ranging from cell cultures to animal models. Unlike C-13 and N-15, P-31-phosphorus is a naturally abundant and NMR active isotope that does not require an external supplemental source. To date, P-31 NMR has seen limited usage in metabolomics because of a lack of reference spectra, difficulties in sample preparation, and an absence of two-dimensional (2D) NMR experiments, but P-31 NMR has the potential of expanding the coverage of the metabolome by detecting phosphorus-containing metabolites. Phosphorylated metabolites regulate key cellular processes, serve as a surrogate for intracellular pH conditions, and provide a measure of a cell's metabolic energy and redox state, among other processes. Thus, incorporating P-31 NMR into a metabolomics investigation will enable the detection of these key cellular processes. To facilitate the application of P-31 NMR in metabolomics, we present a unified protocol that allows for the simultaneous and efficient detection of H-1-C-13-, N-15-, and P-31-labeled metabolites. The protocol includes the application of a 2D H-1-P-31 HSQC-TOCSY experiment to detect P-31-labeled metabolites from heterogeneous biological mixtures, methods for sample preparation to detect H-1-, C-13-, N-15-, and P-31-labeled metabolites from a single NMR sample, and a data set of one-dimensional (1D) P-31 NMR and 2D H-1-P-31 HSQC-TOCSY spectra of 38 common phosphorus-containing metabolites to assist in metabolite assignments.