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. ¹³ C-labeled metabolite tracers or unlabeled ¹ H-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 ¹⁵ N-labeled metabolite tracer experiments that may also be combined with routine ¹³ C tracer experiments to simultaneously detect both ¹⁵ N- and ¹³ C-labeled metabolites in metabolic samples. A database consisting of 2D ¹ H- ¹⁵ N HSQC natural-abundance spectra of 50 nitrogen-containing metabolites are also presented to facilitate the assignment of ¹⁵ N-labeled metabolites. The methodology is demonstrated by labeling Escherichia coli and Staphylococcus aureus metabolomes with ¹⁵ N ₁ -ammonium chloride, ¹⁵ N ₄ -arginine, and ¹³ C ₂ -acetate. Efficient ¹⁵ N and ¹³ C metabolite labeling and identification were achieved utilizing standard cell culture and sample preparation protocols.