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 N1 -ammonium chloride, 15 N4 -arginine, and 13 C2 -acetate. Efficient 15 N and 13 C metabolite labeling and identification were achieved utilizing standard cell culture and sample preparation protocols.