Nuclear Magnetic Resonance (NMR) spectroscopy is a major analytical method for metabolomics, which deals with the high-throughput measurement of the largest possible number of metabolites in a broad variety of biological systems. However, NMR can only detect 50-200 major metabolites out of thousands in the human metabolome, thus potentially hiding significant biomolecular variability, and limiting the discovery of biomarkers to the most concentrated ones. MetaHyp ambitions to improve the sensitivity of NMR metabolomics by two orders of magnitude, thus providing access to a significant fraction of the metabolome which is currently inaccessible to NMR. This will be achieved by developing and optimizing novel hyperpolarized NMR approaches, fully integrated into metabolomics workflows. More specifically, MetaHyp will develop original dissolution dynamic nuclear polarization (d-DNP) protocols and NMR pulse sequences for hyperpolarized 1H and 13C NMR of metabolite mixtures, addressing the sensitivity and throughput bottlenecks that currently limit the application of d-DNP to metabolomics. Methodological developments will include the combination of a cryogen-consumption free d-DNP instrument with a fast and repeatable dissolution and transfer system, as well as parallel detection NMR pulse sequences, tailored solvent suppression methods, and ultrafast 2D NMR experiments. These methods will be integrated into a complete metabolomics workflow, from sample preparation to data analysis, including metabolite quantitation and identification strategies. This approach will be applied to a clinical study, in order to demonstrate its relevance for real-life applications, through the ability to separate different classes of samples, to benchmark their performance versus conventional metabolomics, and ultimately assess their ability to highlight biomarkers that were not accessible to NMR before.
