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.

A Pipeline for Making 31P NMR Accessible for Small- and Large-Scale Lipidomics Studies

“…It also adds to the increasing use of phosphorus NMR in metabolomics [26]. The increasing desire and reliance upon lipidomics and metabolomics data for answering biological questions creates a demand for a choice of finely-drawn methods, not only for a straightforward answer as to the composition of a system but also approaches for verification and validation of data to increase confidence in the omics data acquired.…”

Metabolomic Approaches to Study Chemical Exposure-Related Metabolism Alterations in Mammalian Cell Cultures

“…Carbon-13 NMR is impractical for high-throughput metabolomics NMR analyses due to the low natural abundance (1.1%) of carbon-13 isotopes in biological material [ 81 ]. Phosphorus-31 NMR can be exploited to study intracellular energy states, yet 31P signals from phosphorylated compounds shift heavily upon sample conditions, making signal assignment tedious [ 82 ]. Due to an intrinsic lower sensitivity and scarce natural abundance, nitrogen-15 studies rely on isotopic labeling schemes for targeted pathways analysis.…”

New software tools, databases, and resources in metabolomics: updates from 2020

“…Using with Raman spectroscopy, followed by stimulated Raman scattering (SRS) microscopy and Raman-guided subcellular pharmaco-metabolomics in metastatic melanoma cells revealed intracellular lipid droplets that helped identify a previously unknown susceptibility of lipid mono-unsaturation within de-differentiated mesenchymal cells with innate resistance to BRAF inhibition (Du et al 2020 ). Application of 31 P NMR was shown to hold potential of expanding the coverage of the metabolome by detecting phosphorus-containing metabolites (Bhinderwala et al 2020 ).…”