Stable isotopes are routinely employed by NMR metabolomics to highlight specific metabolic processes and to monitor pathway flux. (13)C-carbon and (15)N-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 (13)C and (15)N, (31)P-phosphourous is a naturally abundant and NMR active isotope that doesn’t require an external supplemental source. To date, (31)P 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, (31)P NMR has the potential of expanding the coverage of the metabolome by detecting phosphorous-containing metabolites. Phosphorylated metabolites regulate key cellular processes, serve as a surrogate for intracellular pH conditions, and provides a measure of a cell’s metabolic energy and redox state, among other processes. Thus, incorporating (31)P NMR into a metabolomics investigation will enable the detection of these key cellular processes. To facilitate the application of (31)P NMR in metabolomics, we present a unified protocol that allows for the simultaneous and efficient detection of (1)H-, (13)C-, (15)N- and (31)P-labeled metabolites. The protocol includes the application of a 2D (1)H-(31)P HSQC-TOCSY experiment to detect (31)P-labeled metabolites from heterogeneous biological mixtures, methods for sample preparation to detect (1)H-, (13)C-, (15)N- and (31)P-labeled metabolites from a single NMR sample, and a dataset of one-dimensional (1D) (31)P NMR and 2D (1)H-(31)P HSQC-TOCSY spectra of 38 common phosphorus-containing metabolites to assist in metabolite assignments.
