Phosphorus NMR and Its Application to Metabolomics.

ABSTRACT: Stable isotopes are routinely employed by NMR metabolomics to highlight specific metabolic processes and to monitor pathway flux. ¹³C-carbon and ¹⁵N-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 ¹³C and ¹⁵N, ³¹P-phosphorus is a naturally abundant and NMR active isotope that does not require an external supplemental source. To date, ³¹P 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 ³¹P 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 ³¹P NMR into a metabolomics investigation will enable the detection of these key cellular processes. To facilitate the application of ³¹P NMR in metabolomics, we present a unified protocol that allows for the simultaneous and efficient detection of ¹H-, ¹³C-, ¹⁵N-, and ³¹P-labeled metabolites. The protocol includes the application of a 2D ¹H-³¹P HSQC-TOCSY experiment to detect ³¹P-labeled metabolites from heterogeneous biological mixtures, methods for sample preparation to detect ¹H-, ¹³C-, ¹⁵N-, and ³¹P-labeled metabolites from a single NMR sample, and a data set of one-dimensional (1D) ³¹P NMR and 2D ¹H-³¹P HSQC-TOCSY spectra of 38 common phosphorus-containing metabolites to assist in metabolite assignments.

SUBMITTER: Bhinderwala F 

PROVIDER: S-EPMC8327684 | biostudies-literature |

REPOSITORIES: biostudies-literature