NMR-Based Stable Isotope Tracing of Cancer Metabolism

NMR is widely used for metabolite profiling (metabolomics, metabonomics) particularly of various readily obtainable biofluids such as plasma and urine. It is especially valuable for stable isotope tracer studies to track metabolic pathways under control or perturbed conditions in a wide range of cell models as well as animal models and human subjects. NMR has unique properties for utilizing stable isotopes to edit or simplify otherwise complex spectra acquired in vitro and in vivo, while quantifying the level of enrichment at specific atomic positions in various metabolites (i.e., isotopomer distribution analysis). In this protocol, we give an overview with specific protocols for NMR-based stable isotope-resolved metabolomics, or SIRM, with a workflow from administration of isotope-enriched precursors, via sample preparation through to NMRNuclear magnetic resonance (NMR) data collection and reduction. We focus on indirect detection of common NMR-active stable isotopesIsotopes including ¹³C, ¹⁵N, ³¹P, and ²H, using a variety of ¹H-based two-dimensional experiments. We also include the application and analyses of multiplex tracer experiments.