Expanding the Coverage of the Metabolome with Nitrogen-Based NMR

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.