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Publications

Journal Article Epublish

PubMed: 40443893 DOI: 10.1021/jacsau.5c00234

Enhanced Standard Operating Procedures for 31P NMR-Based Metabolomics in Tissue Extracts.

JACS Au 5(5):2285-2293

Authors: Sara Martin-Ramos, Jon Bilbao, Tammo Diercks, José M Mato, Ganeko Bernardo-Seisdedos, Oscar Millet

Affiliations

  • Precision Medicine and Metabolism Laboratory, CIC bioGUNE, Basque Research and Technology Alliance, Parque Tecnológico de Bizkaia, Ed. 800, 48160 Derio, Spain.
  • NMR Platform, CIC bioGUNE, Basque Research and Technology Alliance, Parque Tecnológico de Bizkaia, Ed. 800, 48160 Derio, Spain.
  • Precision Medicine and Metabolism Laboratory, CIC bioGUNE, Basque Research and Technology Alliance, Parque Tecnológico de Bizkaia, Ed. 800, 48160 Derio, Spain. CIBERehd, Instituto de Salud Carlos III, Ctra. de Pozuelo, 28, 28222 Majadahonda, Spain.
  • Department of Medicine, Faculty of Health Sciences, University of Deusto, Unibertsitate Etorb. 24, 48007 Bilbao, Spain.
  • ATLAS Molecular Pharma S. L. Parque Tecnológico de Bizkaia, Ed. 800, 48160 Derio, Spain.
  • CIBERehd, Instituto de Salud Carlos III, Ctra. de Pozuelo, 28, 28222 Majadahonda, Spain.

Abstract

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 D 2O) 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.

MeSH

Buffers, Metabolomics, Tissue Extracts, Tissues