Enhanced standard operating procedures for 31P NMR-based metabolomics in tissue extracts
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2025-04-13
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American Chemical Society
Resumen
Phosphorylated metabolites, here referred to as phosphometabolites, are sufficiently abundant and widely distributed to provide a condensed representation of metabolism that can be readily accessed through NMR spectroscopy. This study addresses the challenge of precisely quantifying phosphometabolites via quantitative 31P NMR from tissue extracts. We optimized standard operating procedures for enhanced spectral resolution, signal intensity, and accuracy. By amply evaluating solvent and buffer conditions, reference compounds, and paramagnetic relaxation enhancers, we identified optimal conditions for metabolite analysis, including the use of trimethylphosphine oxide for accurate signal referencing due to its short T1 relaxation time and minimal offset and glycine buffer (in D2O) at pD 9.5, where virtually invariant 31P signal frequencies and sample osmolarities are observed, along with maximal NMR detection sensitivity and temperature stability of the pH. These methodological advancements significantly improve the reliability and reproducibility of phosphometabolite characterization, allowing the assignment of up to 60 independent signals in the one-dimensional (1D) 31P spectrum (of a total of 94 peaks), that resulted in the proper quantification of 44 phosphometabolites from different tissular samples.
Palabras clave
31P NMR
Gadoteridol
Glycine buffer
Metabolites
Phosphorylated metabolites
SOP
Tissue analysis
Gadoteridol
Glycine buffer
Metabolites
Phosphorylated metabolites
SOP
Tissue analysis
Descripción
Materias
Cita
Martin-Ramos, S., Bilbao, J., Diercks, T., Mato, J. M., Bernardo-Seisdedos, G., & Millet, O. (2025). Enhanced standard operating procedures for 31P NMR-based metabolomics in tissue extracts. JACS Au, 5(5), 2285-2293. https://doi.org/10.1021/JACSAU.5C00234