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Expanding the Coverage of the Metabolome with Nitrogen-Based NMR
- Bhinderwala, Fatema, Lonergan, Samantha, Woods, Jade, Zhou, Chunyi, Fey, Paul D., Powers, Robert
- Analytical chemistry 2018 v.90 no.7 pp. 4521-4528
- Escherichia coli, Staphylococcus aureus, carbon, cell culture, chlorides, databases, human diseases, isotope labeling, metabolism, metabolites, metabolome, metabolomics, nitrogen, nuclear magnetic resonance spectroscopy, protocols, stable isotopes
- Isotopically labeling a metabolite and tracing its metabolic fate has provided invaluable insights about the role of metabolism in human diseases in addition to a variety of other issues. ¹³C-labeled metabolite tracers or unlabeled ¹H-based NMR experiments are currently the most common application of NMR to metabolomics studies. Unfortunately, the coverage of the metabolome has been consequently limited to the most abundant carbon-containing metabolites. To expand the coverage of the metabolome and enhance the impact of metabolomics studies, we present a protocol for ¹⁵N-labeled metabolite tracer experiments that may also be combined with routine ¹³C tracer experiments to simultaneously detect both ¹⁵N- and ¹³C-labeled metabolites in metabolic samples. A database consisting of 2D ¹H–¹⁵N HSQC natural-abundance spectra of 50 nitrogen-containing metabolites are also presented to facilitate the assignment of ¹⁵N-labeled metabolites. The methodology is demonstrated by labeling Escherichia coli and Staphylococcus aureus metabolomes with ¹⁵N₁-ammonium chloride, ¹⁵N₄-arginine, and ¹³C₂-acetate. Efficient ¹⁵N and ¹³C metabolite labeling and identification were achieved utilizing standard cell culture and sample preparation protocols.