Hyperpolarized NMR Metabolomics at Natural 13C Abundance.

Metabolomics plays a pivotal role in systems biology, and NMR is a central tool with high precision and exceptional resolution of chemical information. Most NMR metabolomic studies are based on 1H 1D spectroscopy, severely limited by peak overlap. 13C NMR benefits from a larger signal dispersion but is barely used in metabolomics due to ca. 6000-fold lower sensitivity. We introduce a new approach, based on hyperpolarized 13C NMR at natural abundance, that circumvents this limitation. A new untargeted NMR-based metabolomic workflow based on dissolution dynamic nuclear polarization (d-DNP) for the first time enabled hyperpolarized natural abundance 13C metabolomics. Statistical analysis of resulting hyperpolarized 13C data distinguishes two groups of plant (tomato) extracts and highlights biomarkers, in full agreement with previous results on the same biological model. We also optimize parameters of the semiautomated d-DNP system suitable for high-throughput studies.

Mass spectrometry methods for intrinsically disordered proteins.

In the last ten years mass spectrometry has emerged as a powerful biophysical technique capable of providing unique insights into the structure and dynamics of proteins. Part of this explosion in use involves investigations of the most recently 'discovered' subset of proteins: the so-called 'Intrinsically Disordered' or 'Natively Unstructured' proteins. A key advantage of the use of mass spectrometry to study intrinsically disordered proteins (IDPs) is its ability to test biophysical assertions made about why they differ from structured proteins. For example, from the charge state distribution presented by a protein following nano-electrospray (n-ESI) it is possible to infer the range of conformations present in solution and hence the extent of disorder; n-ESI is highly sensitive to the degree of folding at the moment of transfer from the liquid to the gas phase. The combination of mass spectrometry with ion mobility (IM-MS) provides rotationally averaged collision cross-sections of molecular ions which can be correlated with conformation; this too can be applied to IDPs. Another feature which can be monitored by IM-MS is the tendency of disordered proteins to form amyloid fibrils, the protein aggregates involved in the onset of neurodegenerative diseases such as Parkinson's and Alzheimer's. IM-MS provides a useful insight into events that occur during the early stages of aggregation including delineating the structure of the monomer, identifying oligomer distributions, and revealing mechanistic details of the aggregation process. Here we will review the use of MS and IM-MS to study IDPs using examples from our own and other laboratories.

Measuring absolute spin polarization in dissolution-DNP by Spin PolarimetrY Magnetic Resonance (SPY-MR).

Dynamic nuclear polarization at 1.2 K and 6.7 T allows one to achieve spin temperatures on the order of a few millikelvin, so that the high-temperature approximation (ΔE

Hyperpolarized Water to Study Protein-Ligand Interactions.

The affinity between a chosen target protein and small molecules is a key aspect of drug discovery. Screening by popular NMR methods such as Water-LOGSY suffers from low sensitivity and from false positives caused by aggregated or denatured proteins. This work demonstrates that the sensitivity of Water-LOGSY can be greatly boosted by injecting hyperpolarized water into solutions of proteins and ligands. Ligand binding can be detected in a few seconds, whereas about 30 min is usually required without hyperpolarization. Hyperpolarized water also enhances proton signals of proteins at concentrations below 20 µM so that one can verify in a few seconds whether the proteins remain intact or have been denatured.