Journal
ANALYTICAL CHEMISTRY
Volume 90, Issue 7, Pages 4521-4528Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.7b04922
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Funding
- National Science Foundation [1660921]
- Redox Biology Center (NIGMS) [P30 GM103335]
- Nebraska Center for Integrated Biomolecular Communication (NIGMS) [P20 GM113126]
- National Institutes of Health (NIAID) [P01 AI083211]
- National Institutes of Health [RR015468-01]
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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. C-13-labeled metabolite tracers or unlabeled H-1-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 N-15-labeled metabolite tracer experiments that may also be combined with routine C-13 tracer experiments to simultaneously detect both N-15- and C-13-labeled metabolites in metabolic samples. A database consisting of 2D H-1-N-15 HSQC natural-abundance spectra of SO nitrogen-containing metabolites are also presented to facilitate the assignment of N-15-labeled metabolites. The methodology is demonstrated by labeling Escherichia coli and Staphylococcus aureus metabolomes with N-15(1)-ammonium chloride, N-15(4)-arginine, and C-13(2)-acetate. Efficient N-15 and C-13 metabolite labeling and identification were achieved utilizing standard cell culture and sample preparation protocols.
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