Journal
ANALYTICAL CHEMISTRY
Volume 92, Issue 14, Pages 9536-9545Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.0c00591
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Funding
- National Science Foundation [1660921]
- Redox Biology Center (NIGMS) [P30 GM103335]
- Nebraska Center for Integrated Biomolecular Communication (NIGMS) [P20 GM113126]
- National Institutes of Health [RR015468-01]
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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.
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