Abhinav Dubey

A fast NMR method for resonance assignments: application to metabolomics.

We present a new method for rapid NMR data acquisition and assignments applicable to unlabeled (12C) or 13C-labeled biomolecules/organic molecules in general and metabolomics in particular. The method involves the acquisition of three two dimensional (2D) NMR spectra simultaneously using a dual receiver system. The three spectra, namely: (1) G-matrix Fourier transform (GFT) (3,2)D [13C, 1H] HSQC–TOCSY, (2) 2D 1H–1H TOCSY and (3) 2D 13C–1H HETCOR are acquired in a single experiment and provide mutually complementary information to completely assign individual metabolites in a mixture. The GFT (3,2)D [13C, 1H] HSQC–TOCSY provides 3D correlations in a reduced dimensionality manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete 1H and 13C assignments of a mixture of 21 unlabeled metabolites corresponding to a medium used in assisted reproductive technology. Taken together, the experiments provide time gain of order of magnitudes compared to the conventional data acquisition methods and can be combined with other fast NMR techniques such as non-uniform sampling and covariance spectroscopy. This provides new avenues for using multiple receivers and projection NMR techniques for high-throughput approaches in metabolomics.

Pattern Recognition-Based Approach for Identifying Metabolites in Nuclear Magnetic Resonance-Based Metabolomics.

Identification and assignments of metabolites is an important step in metabolomics and is necessary for the discovery of new biomarkers. In nuclear magnetic resonance (NMR) spectroscopy-based studies, the conventional approach involves a database search, wherein chemical shifts are assigned to specific metabolites by use of a tolerance limit. This is inefficient because deviation in chemical shifts associated with pH or temperature variations, as well as missing peaks, impairs a robust comparison with the database. We propose here a novel method based on matching the pattern of peaks rather than absolute tolerance thresholds, using a combination of geometric hashing and similarity scoring techniques. Tests with 719 metabolites from the Human Metabolome Database (HMDB) show that 100% of the metabolites can be assigned correctly when accurate data are available. A high success rate is obtained even in the presence of large chemical shift deviations such as 0.5 ppm in (1)H and 3 ppm in (13)C and missing peaks (up to 50%), compared to nearly no assignments obtained under these conditions with existing methods that employ a direct database search approach. The method was evaluated on experimental data on a mixture of 16 metabolites at eight different combinations of pH and temperature conditions. The pattern recognition approach thus helps in identification and assignment of metabolites independent of the pH, temperature, and ionic strength used, thereby obviating the need for spectral calibration with internal or external standards.

Rapid NMR Assignments of Proteins by Using Optimized Combinatorial Selective Unlabeling

A new approach for rapid resonance assignments in proteins based on amino acid selective unlabeling is presented. The method involves choosing a set of multiple amino acid types for selective unlabeling and identifying specific tripeptides surrounding the labeled residues from specific 2D NMR spectra in a combinatorial manner. The methodology directly yields sequence specific assignments, without requiring a contiguously stretch of amino acid residues to be linked, and is applicable to deuterated proteins. We show that a 2D [15N,1H] HSQC spectrum with two 2D spectra can result in ∼50 % assignments. The methodology was applied to two proteins: an intrinsically disordered protein (12 kDa) and the 29 kDa (268 residue) α‐subunit of Escherichia coli tryptophan synthase, which presents a challenging case with spectral overlaps and missing peaks. The method can augment existing approaches and will be useful for applications such as identifying active‐site residues involved in ligand binding, phosphorylation, or protein–protein interactions, even prior to complete resonance assignments.

SIRT6 deacetylase transcriptionally regulates glucose metabolism in heart

Sirtuins are a family of enzymes, which govern a number of cellular processes essential for maintaining physiological balance. SIRT6, a nuclear sirtuin, is implicated in the development of metabolic disorders. The role of SIRT6 in regulation of cardiac metabolism is unexplored. Although glucose is not the primary energy source of heart, defects in glucose oxidation have been linked to heart failure. SIRT6+/− mice hearts exhibit increased inhibitory phosphorylation of PDH subunit E1α. SIRT6 deficiency enhances FoxO1 nuclear localization that results in increased expression of PDK4. We show that SIRT6 transcriptionally regulates the expression of PDK4 by binding to its promoter. SIRT6+/− hearts show accumulation of lactate, indicating compromised mitochondrial oxidation. SIRT6 deficiency results in decreased oxygen consumption rate and concomitantly lesser ATP production. Mechanistically, SIRT6 deficiency leads to increased FoxO1‐mediated transcription of PDK4. Our findings establish a novel link between SIRT6 and cardiac metabolism, suggesting a protective role of SIRT6 in maintaining cardiac homeostasis.

Rapid characterization of hydrogen exchange in proteins.

Kinetics and thermodynamics of amide hydrogen exchange in proteins can be investigated with two-dimensional (13)CO-(15)N NMR correlation experiments. The spectra are acquired with high resolution and sensitivity. A single type of experiment on one sample serves to characterize hydrogen-deuterium fractionation factors and hydrogen-exchange rates that span three orders of magnitude.

Ultra broadband NMR spectroscopy using multiple rotating frame technique.

The paper describes the design of broadband excitation and inversion pulses by method of multiple rotating frame technique. The ideal situation for perfect excitation and inversion is to have no chemical shift offset and thereby everything on resonance. However, when chemical shifts span a wide range, this condition is not realized. We achieve this condition using a multiply modulated rf-field, whose effect can be understood by progressing into multiple frames. As we progress through the frames, the ratio of chemical shift dispersion to strength of static rf-field decreases, resulting in a final frame, where there is negligible chemical shift as compared to the effective rf-field and we get good excitation and inversion. Increasing the number of frames, increases excitation bandwidth and the ratio of bandwidth to rms excitation amplitude. We show, in principle, it is possible to excite arbitrary large bandwidth for a given rms rf-amplitude by increasing the number of frames. The time of excitation increases linearly with the bandwidth when we keep the rms rf-amplitude constant. Experimental demonstration of proposed method is presented on (1)H excitation over a bandwidth of 52 kHz with a rms amplitude of 10 kHz. Increasing the frames increases excitation bandwidth for same rms amplitude of 10 kHz. Experimental spectra obtained from 100%(13)C labeled arginine shows uniform excitation over the entire carbon spectra, obtained with a 8-frame pulse sequence.

Rapid identification of amino acid types in proteins using phase modulated 2D HN(CACB) and 2D HN(COCACB)

We present a simple approach to rapidly identify amino acid types in proteins from a 2D spectrum. The method is based on the fact that (13)C(β) chemical shifts of different amino acid types fall in distinct spectral regions. By evolving the (13)C chemical shifts in the conventional HNCACB or HN(CO)CACB type experiment for a single specified delay period, the phase of the cross peaks of different amino acid residues are modulated depending on their (13)C(β) shift values. Following this specified evolution period, the 2D HN projections of these experiments are acquired. The (13)C evolution period can be chosen such that all residues belonging to a given set of amino acid types have the same phase pattern (positive or negative) facilitating their identification. This approach does not require the preparation of any additional samples, involves the analysis of 2D [(15)N-(1)H] HSQC-type spectra obtained from the routinely used triple resonance experiments with minor modifications, and is applicable to deuterated proteins. The method will be useful for quick assignment of signals that shift during ligand binding or in combination with selective labeling/unlabeling approaches for identification of amino acid types to aid the sequential assignment process.

Simultaneous acquisition of three NMR spectra in a single experiment for rapid resonance assignments in metabolomics

AbstractNMR-based approach to metabolomics typically involves the collection of two-dimensional (2D) heteronuclear correlation spectra for identification and assignment of metabolites. In case of spectral overlap, a 3D spectrum becomes necessary, which is hampered by slow data acquisition for achieving sufficient resolution. We describe here a method to simultaneously acquire three spectra (one 3D and two 2D) in a single data set, which is based on a combination of different fast data acquisition techniques such as G-matrix Fourier transform (GFT) NMR spectroscopy, parallel data acquisition and non-uniform sampling. The following spectra are acquired simultaneously: (1) 13C multiplicity edited GFT (3,2)D HSQC-TOCSY, (2) 2D [ 1H- 1H] TOCSY and (3) 2D [ 13C- 1H] HETCOR. The spectra are obtained at high resolution and provide high-dimensional spectral information for resolving ambiguities. While the GFT spectrum has been shown previously to provide good resolution, the editing of spin systems based on their CH multiplicities further resolves the ambiguities for resonance assignments. The experiment is demonstrated on a mixture of 21 metabolites commonly observed in metabolomics. The spectra were acquired at natural abundance of 13C. This is the first application of a combination of three fast NMR methods for small molecules and opens up new avenues for high-throughput approaches for NMR-based metabolomics. Graphical AbstractWe describe here a first application of a combination of different fast NMR methods to simultaneously acquire three spectra in a single data set. The method combines G-matrix Fourier transform (GFT) NMR spectroscopy, parallel data acquisition and non-uniform sampling. This opens up new avenues for high-throughput approaches in metabolomics.

Phase modulated 2D HSQC-TOCSY for unambiguous assignment of overlapping spin systems

We present a new method that allows one to unambiguously resolve overlapping spin systems often encountered in biomolecular systems such as peptides and proteins or in samples containing a mixture of different molecules such as in metabolomics. We address this problem using the recently proposed phase modulation approach. By evolving the 1H chemical shifts in a conventional two dimensional (2D) HSQC-TOCSY experiment for a fixed delay period, the phase/intensity of set of cross peaks belonging to one spin system are modulated differentially relative to those of its overlapping counterpart, resulting in their discrimination and recognition. The method thus accelerates the process of identification and resonance assignment of individual compounds in complex mixtures. This approach facilitated the assignment of molecules in the embryo culture medium used in human assisted reproductive technology.