¹³C NMR Metabolomics: INADEQUATE Network Analysis

Abstract

The many advantages of 13C NMR are often overshadowed by its intrinsically low sensitivity. Given that carbon makes up the backbone of most biologically relevant molecules, 13C NMR offers a straightforward measurement of these compounds. Two-dimensional 13C-13C correlation experiments like INADEQUATE (incredible natural abundance double quantum transfer experiment) are ideal for the structural elucidation of natural products and have great but untapped potential for metabolomics analysis. We demonstrate a new and semi-automated approach called INETA (INADEQUATE network analysis) for the untargeted analysis of INADEQUATE datasets using an in silico INADEQUATE database. We demonstrate this approach using isotopically labeled Caenorhabditis elegans mixtures.

The future of NMR-based metabolomics

“…Small high-temperature superconducting coils can maximize the signal per sample mass: a 13 C-optimized 1.5-mm high temperature superconducting NMR probe has enabled novel 13 C NMR studies of natural products [47], and this has been followed up with a 1 H- 13 C dual-optimized NMR probe based on double-tuned high temperature superconducting resonators [48]. These probes take advantage of the excellent peak dispersion of 13 C spectra [49], which can be augmented by further 2D 13 C- 13 C experiments, such as INADEQUATE [50]…”

Two elephants in the room

“…These issues can be largely overcome by using reference 2D NMR spectra for each compound or methods that allow one to subtract the mixing time effects, as is the case in the HSQC 0 approach [ 28 ]. Alternatively, one can use experiments that are relatively immune to small variations in J-coupling constants as was shown for 13 C- 13 C CT-TOCSY of uniformly 13 C-labeled mixtures [ 29 ] provided that the carbon-backbone topology is known, which can be readily extracted from the same 2D NMR experiment [ 30 ] or from an Incredible Natural Abundance Double Quantum Transfer Experiment (INADEQUATE)-type experiment [ 31 ]. A main limitation of 2D NMR is the need for longer measurement times.…”

Knowns and unknowns in metabolomics identified by multidimensional NMR and hybrid MS/NMR methods

“…[31] A similar strategy has been successfully applied to fully 13 C-labeled C. elegans cell lysate by using the 2D 13 C- 13 C INADEQUATE experiment, which correlates pairs of directly J-coupled 13 C spins in the metabolites. [32] Determination of the carbon backbone topology of each spin system of a molecule represents the first step for structure elucidation. Next, one needs to connect multiple backbone topologies of each molecule by using long-range correlation experiments; moreover, one has to identify the (non-carbon) substituents (functional groups) that are attached to the carbon skeleton.…”