NMR Acquisition Research Articles

Optimising in-cell NMR acquisition for nucleic acids.

Understanding the structure and function of nucleic acids in their native environment is crucial to structural biology and one focus of in-cell NMR spectroscopy. Many challenges hamper in-cell NMR in human cell lines, e.g. sample decay through cell death and RNA degradation. The resulting low signal intensities and broad line widths limit the use of more complex NMR experiments, reducing the possible structural and dynamic information that can be extracted. Here, we optimize the detection of imino proton signals, indicators of base-pairing and therefore secondary structure, of a double-stranded DNA oligonucleotide in HeLa cells, using selective excitation. We demonstrate the reproducible quantification of in-cell selective longitudinal relaxation times (selT1), which are reduced compared to the in vitro environment, as a result of interactions with the complex cellular environment. By measuring the intracellular selT1, we optimize the existing proton pulse sequences, and shorten measurement time whilst enhancing the signal gained per unit of time. This exemplifies an advantage of selective excitation over conventional methods like jump-return water suppressionfor in-cell NMR. Furthermore, important experimental controls are discussed, including intracellular quantification, supernatant control measurements, as well as the processing of lowly concentrated in-cell NMR samples. We expect that robust and fast in-cell NMR experiments of nucleic acids will facilitate the study of structure and dynamics and reveal their functional correlation.

Optimizing NMR fragment-based drug screening for membrane protein targets

NMR spectroscopy has played a pivotal role in fragment-based drug discovery by coupling detection of weak ligand-target binding with structural mapping of the binding site. Fragment-based screening by NMR has been successfully applied to many soluble protein targets, but only to a limited number of membrane proteins, despite the fact that many drug targets are membrane proteins. This is partly because of difficulties preparing membrane proteins for NMR—especially human membrane proteins—and because of the inherent complexity associated with solution NMR spectroscopy on membrane protein samples, which require the inclusion of membrane-mimetic agents such as micelles, nanodiscs, or bicelles. Here, we developed a generalizable protocol for fragment-based screening of membrane proteins using NMR. We employed two human membrane protein targets, both in fully protonated detergent micelles: the single-pass C-terminal domain of the amyloid precursor protein, C99, and the tetraspan peripheral myelin protein 22 (PMP22). For both we determined the optimal NMR acquisition parameters, protein concentration, protein-to-micelle ratio, and upper limit to the concentration of D6-DMSO in screening samples. Furthermore, we conducted preliminary screens of a plate-format molecular fragment mixture library using our optimized conditions and were able to identify hit compounds that selectively bound to the respective target proteins. It is hoped that the approaches presented here will be useful in complementing existing methods for discovering lead compounds that target membrane proteins.

Current trends in ŒNO-NMR based metabolomics

Present work discusses strengths and limitations of two Nuclear Magnetic Resonance outliers obtained with a water-to-ethanol solvent multi pre saturation acquisition method, recently included in the Compendium of International Methods of Analysis of Wines and Musts, published as OIV-MA-AS316-01, and their accuracy for metabolomics analysis. Furthermore, it is also presented an alternative to produce more discriminant and sensitive NMR data matrices for metabolomics studies, comprising the use of a novel NMR acquisition strategy in wines, the double pulsed-field gradient echo (DPFGE) NMR scheme, with a refocusing band-selective uniform-response pure-phase selective pulse, for a selective excitation of the 5-10 ppm chemical shift range of wine samples, that reveals novel broad aromatic 1H resonances, directly associated to complex polyphenols. Both aromatics and full binned OIV-MA-AS316-01,as well as the selective 5-10 ppm DPFGE NMR outliers were statistically analyzed with diverse non-supervised Principal Component Analysis (PCA) and supervised Partial Least Squares -Discriminant Analysis (PLS-DA), sparse (sPLS-DA) least squares- discriminant analysis, and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Supervised multivariate statistical analysis of DPFGE and aromatics’ binned OIV-MA-AS316-01NMR data have shown their robustness to broadly discriminate geographical origins and narrowly differentiate between different fermentation schemes of wines from identical variety and region.

Fast and accurate diffusion NMR acquisition in continuous flow.

FlowNMR spectroscopy has become a popular and powerful technique for online reaction monitoring. DOSY NMR is an established technique for obtaining information about diffusion rates and molecular size on static samples. This work extends the FlowNMR toolbox to include FlowDOSY based on convection compensation and use of a low-pulsation pump or flow effect correction, allowing accurate and precise diffusion coefficients to be obtained at flow rates up to 4.0 mL min-1 in less than 5 minutes.

In vivo hyperCEST imaging: Experimental considerations for a reliable contrast.

HyperCEST contrast relies on the reduction of the solvent signal after selective saturation of the solute magnetization. The scope of this work is to outline the experimental conditions needed to obtain a reliable hyperCEST contrast in vivo, where the "solvent" signal (ie, the dissolved-phase signal) may change over time due to the increase in xenon (Xe) accumulation into tissue. Hyperpolarized 129 Xe was delivered to mice at a constant volume and rate using a mechanical ventilator, which triggered the saturation, excitation, and acquisition of the MR signal during the exhale phase of the breath cycle-either every breath or every 2, 3, or 4 breaths. Serial Z-spectra and hyperCEST images were acquired before and after a bolus injection of cucurbit[6]uril to assess possible signal fluctuations and instabilities. The intensity of the dissolved-phase Xe signal was observed to first increase immediately after the beginning of the hyperpolarized gas inhalation and NMR acquisition, and then decrease before reaching a steady-state condition. Once a steady-state dissolved-phase magnetization was established, a reliable hyperCEST contrast, exceeding 40% signal reduction, was observed. A reliable hyperCEST contrast can only be obtained after establishing a steady-state dissolved phase 129 Xe magnetization. Under stable physiological conditions, a steady-state dissolved-phase Xe magnetization is only achieved after a series of Xe inhalations and RF excitations, and it requires synchronization of the breathing rate with the MR acquisition.

Quantitative 31P NMR Analysis of Lignins and Tannins.

The development of sustainable biorefinery products is confronted, among others, with the challenge of lignin and tannin valorization. These abundant, renewable aromatic biopolymers have not been widely exploited due to their inherent structural complexity and high degrees of variability and species diversity. The lack of a defined primary structure for these polyphenols is further compounded with complex chemical alterations induced during processing, eventually imparting a large variety of structural features of extreme significance for any further utilization efforts. Consequently, a protocol for the rapid, simple, and unequivocal identification and quantification of the various functional groups present in natural polyphenols, is a fundamental prerequisite for understanding and accordingly tailor their reactivity and eventual utility. Quantitative 31P NMR offers the opportunity to rapidly and reliably identify unsubstituted, o-mono substituted, and o-disubstituted phenols, aliphatic OHs, and carboxylic acid moieties in lignins and tannins with broad application potential. The methodology consists of an in situ quantitative lignin or tannin labeling procedure using a suitable 31P containing probe, followed by the acquisition of a quantitative 31P NMR spectrum in the presence of an internal standard. The high natural abundance of the 31P nucleus allows for small amounts of the sample (~30 mg) and short NMR acquisition times (~30-120 min) with well-resolved 31P signals that are highly dependent on the surrounding chemical environment of the labeled OH groups.

On-the-Fly, Sample-Tailored Optimization of NMR Experiments.

NMR experiments, indispensable to chemists in many areas of research, are often run with generic, unoptimized experimental parameters. This approach makes robust and automated acquisition on different samples and instruments extremely challenging. Here, we present NMR-POISE (Parameter Optimization by Iterative Spectral Evaluation), the first demonstration of on-the-fly, sample-tailored, and fully automated optimization of a wide range of NMR experiments. We illustrate how POISE maximizes spectral sensitivity and quality with a diverse set of 1D and 2D examples, ranging from HSQC and NOESY experiments to ultrafast and pure shift techniques. Our Python implementation of POISE has an interface integrated into Bruker's TopSpin software, one of the most widely used platforms for NMR acquisition and automation, allowing NMR optimizations to be run without direct user supervision. We predict that POISE will find widespread usage in academia and industry, where sample-specific and automated experiment optimization is mandatory.

Integrated impedance sensing of liquid sample plug flow enables automated high throughput NMR spectroscopy

A novel approach for automated high throughput NMR spectroscopy with improved mass-sensitivity is accomplished by integrating microfluidic technologies and micro-NMR resonators. A flow system is utilized to transport a sample of interest from outside the NMR magnet through the NMR detector, circumventing the relatively vast dead volume in the supplying tube by loading a series of individual sample plugs separated by an immiscible fluid. This dual-phase flow demands a real-time robust sensing system to track the sample position and velocities and synchronize the NMR acquisition. In this contribution, we describe an NMR probe head that possesses a microfluidic system featuring: (i) a micro saddle coil for NMR spectroscopy and (ii) a pair of interdigitated capacitive sensors flanking the NMR detector for continuous position and velocity monitoring of the plugs with respect to the NMR detector. The system was successfully tested for automating flow-based measurement in a 500 MHz NMR system, enabling high resolution spectroscopy and NMR sensitivity of 2.18 nmol s1/2 with the flow sensors in operation. The flow sensors featured sensitivity to an absolute difference of 0.2 in relative permittivity, enabling distinction between most common solvents. It was demonstrated that a fully automated NMR measurement of nine individual 120 μL samples could be done within 3.6 min or effectively 15.3 s per sample.

Overhauser dynamic nuclear polarization (ODNP)-enhanced two-dimensional proton NMR spectroscopy at low magnetic fields.

The majority of low-field Overhauser dynamic nuclear polarization (ODNP) experiments reported so far have been 1D NMR experiments to study molecular dynamics and in particular hydration dynamics. In this work, we demonstrate the application of ODNP-enhanced 2D J-resolved (JRES) spectroscopy to improve spectral resolution beyond the limit imposed by the line broadening introduced by the paramagnetic polarizing agent. Using this approach, we are able to separate the overlapping multiplets of ethyl crotonate into a second dimension and clearly identify each chemical site individually. Crucial to these experiments is interleaved spectral referencing, a method introduced to compensate for temperature-induced field drifts over the course of the NMR acquisition. This method does not require additional hardware such as a field-frequency lock, which is especially challenging when designing compact systems.

Comparative NMR Metabolomics Profiling between Mexican Ancestral & Artisanal Mezcals and Industrialized Wines to Discriminate Geographical Origins, Agave Species or Grape Varieties and Manufacturing Processes as a Function of Their Quality Attributes.

The oenological industry has benefited from the use of Nuclear Magnetic Resonance (1H-NMR) spectroscopy in combination with Multivariate Statistical Analysis (MSA) as a foodomics tool for retrieving discriminant features related to geographical origins, grape varieties, and further quality controls. Said omics methods have gained such attention that Intergovernmental Organizations and Control Agencies are currently recommending their massive use amongst countries as quality compliances for tracking standard and degradation parameters, fermentation products, polyphenols, amino acids, geographical origins, appellations d’origine contrôlée and type of monovarietal strains in wines. This study presents, for the first time, a 1H-NMR/MSA profiling of industrial Mexican wines, finding excellent statistical features to discriminate between oenological regions and grape varieties with supervised Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA). In a comparative way, it is applied with the 1H-NMR/OPLS-DA workflow for the first time in ancestral and artisanal Mexican mezcals with promising results to discriminate between regions, agave species and manufacturing processes. The central aim of this comparative study is to extrapolate the know-how of wine-omics into the non-professionalized mezcal industry for establishing the NMR acquisition, preprocessing and statistical analysis basis to implement novel, non-invasive and highly reproducible regional, agave species and manufacturing-quality controls.

Investigation of poly(α-methyl styrene) tacticity synthesized by photo-polymerization

In this work, poly(α-methyl styrene) (PAS) was synthesized by free radical photo-polymerization. The tacticity and microstructure of PAS were studied by 13C nuclear magnetic resonance spectroscopy (13C NMR). Assignments of all stereo sequences were carried out at pentad and hexad levels of quaternary aliphatic, quaternary aromatic, and methylene carbons using 13C NMR in deuterated chloroform at 20 °C and 50 °C, respectively. It was found that by increasing the NMR acquisition temperature, higher resolution and higher splitting of the peaks were achieved for all mentioned carbons. Bernoullian and first-order Markov statistics were used for all carbons and the results were compared with experimental data by statistical method. It was shown that Bernoullian statics model fit slightly better than first-order Markov statics model for the assigned sequences. The results indicated that corresponding values of probability (Pm) was equal to 0.234, suggesting that the racemic addition was almost higher than the meso-one. The effect of the deuterated solvent on the peak resolution and the splitting of syndiotactic and isotactic sequences was investigated by replacing deuterated tetrahydrofuran with deuterated chloroform. It was observed that the peak resolution and the splitting in deuterated chloroform are much more than deuterated tetrahydrofuran.

Solid-state 31P NMR mapping of active centers and relevant spatial correlations in solid acid catalysts.

Solid acid catalysts are used extensively in various advanced chemical and petrochemical processes. Their catalytic performance (namely, activity, selectivity, and reaction pathway) mostly depends on their acid properties, such as type (Brønsted versus Lewis), location, concentration, and strength, as well as the spatial correlations of their acid sites. Among the diverse methods available for acidity characterization, solid-state nuclear magnetic resonance (SSNMR) techniques have been recognized as the most valuable and reliable tool, especially in conjunction with suitable probe molecules that possess observable nuclei with desirable properties. Taking 31P probe molecules as an example, both trimethylphosphine (TMP) and trimethylphosphine oxide (TMPO) adsorb preferentially to the acid sites on solid catalysts and thus are capable of providing qualitative and quantitative information for both Brønsted and Lewis acid sites. This protocol describes procedures for (i) the pretreatment of typical solid acid catalysts, (ii) adoption and adsorption of various 31P probe molecules, (iii) considerations for one- and two-dimensional (1D and 2D, respectively) NMR acquisition, (iv) relevant data analysis and spectral assignment, and (v) methodology for NMR mapping with the assistance of theoretical calculations. Users familiar with SSNMR experiments can complete 31P-1H heteronuclear correlation (HETCOR), 31P-31P proton-driven spin diffusion (PDSD), and double-quantum (DQ) homonuclear correlation with this protocol within 2-3 d, depending on the complexity and the accessible acid sites of the solid acid samples.

Classification of the botanical and geographical origins of Chinese honey based on 1H NMR profile with chemometrics

In this paper, we report a newly developed non-target 1H NMR detection associated with chemometrics method to classify the botanical and geographical origins of the monofloral Chinese honey. 1H NMR tests of 218 monofloral honey samples of 8 classes (Acacia, Jujube, Linden, Longan, Orange, Rape, Sunflower, Vitex) collected in 2017–2019 across China were conducted under the optimal sample preparation conditions and NMR acquisition parameters. The whole profiles of NMR spectra instead of individual or partial signals from specific components were processed and extracted, then fed to SIMCA-P to classify the botanical and geographical origins through non-target statistical analysis. For the botanical origins, most of them could be classified clearly according to Principal Component Analysis (PCA) with both R2 and Q2 close to 1. Orthogonal Partial Least Squares Discrimination Analysis (OPLS-DA) model could classify the honey floral types successfully with R2Y and Q2 greater than 0.85. It is found that the integral bin for data extraction has no obvious influence on the classification. For the geographical origins, the classification at different geographical levels (providence and town) could be successfully distinguished by OPLS-DA model. The promising preliminary results with the geographical classification at 40 km level unambiguously demonstrate the application of this NMR-based multi-species non-targeted method for the honey authenticity. Successful result is obtained on a pilot prediction of the geographical classification. Comparing with the methods based on other techniques, the advantages of this reported one are less sample amount needed, simple preparation, short test time, and non-targeted multi-species detection.

Analyses of Short Chain Branches in Polyolefins with Improved 1H NMR Spectroscopy.

Polyolefin microstructures, for example, short chain branching (SCB) and short chain branch distribution (SCBD), have a direct impact on properties and thus ultimately influence end-use applications. The 1H NMR approach to analyze SCB and SCBD is particularly useful when only a limited amount of sample is available, for example, polyolefin film layers or the fractions from polyolefin separation techniques, such as gel permeation chromatography (GPC), crystallization elution fractionation (CEF), high temperature liquid chromatography (HTLC), and thermal gradient interaction chromatography (TGIC). In this paper, we discuss the best approach to find a good decoupling frequency and propose an improved 1H pulse sequence with homonuclear decoupling for better measuring SCB. With this new pulse it is possible to reach a S/N of 10 (level of quantification) for the methyl signal from SCB in an ethylene-hexene copolymer (EH, 3.6 mol % H) in 3.5 min with 0.5 μg of sample. We also show an easy method to calculate SCB/1000C and demonstrate the proper use of heteronuclear single quantum coherence (HSQC) to measure SCB in a complicated system. A very quick approach to examine the presence of a small amount of LDPE in a polyolefin sample is also suggested, which can reduce NMR acquisition time from a couple of days to a few minutes.

Real Time Direct Detection of β – hydroxybutyrate Production in Perfused Mice Livers Using HP DHA

Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH) are increasingly prevalent in the United States. A diagnosis of NAFLD left untreated directly leads to the increase of NASH morbidity which is a result of over‐nutrition, poor diet, lack of exercise, and a sedentary lifestyle. However, NAFLD is typically reversible with a change in lifestyle. NAFLD is a group of common conditions that occur when excess fat accumulates in the liver of those who consume little alcohol. Changes in ketogenesis within the liver has been associated with NAFLD development in high‐fat diet models in mice, and in humans, lower ketogenesis is associated with an increase in hepatic steatosis. Since changes in ketogenesis are associated with different physiological conditions, it would be beneficial if the de novo ketone production can be measured in real‐time.Here, we report the detection of β‐hydroxybutyrate (BHB) in perfused liver using hyperpolarized [2‐13C] dihydroxyacetone (HP‐DHA) and Magnetic Resonance Spectroscopy (MRS). Dihydroxyacetone (DHA) is avidly taken up by the liver and rapidly phosphorylated into dihydroxyacetone phosphate (DHAP). DHAP is then metabolized into (Fig. 1) phosphoenolpyruvate (PEP), glycerol‐3‐phosphate (G3P) and glucose (in fasted mice), representing glycolysis, fatty acid synthesis, and gluconeogenesis, respectively. HP‐DHA injection does not drive BHB production by a mass action effect. Fasting mice show significantly increased ketone production, but a low alanine and lactate signal are observed as gluconeogenesis is increased. BHB is a compelling indicator of rapid flux of carbon skeletons towards ketones. Metabolism of DHA to BHB requires at least 11 steps with most of the metabolites in fasted mice not being observable. This establishes the interplay between pool size and observable HP MR Signal.The chemical shift of BHB makes it a prime target for imaging, as it does not overlap with other resonances. While previous research designs have been too complicated for clinical application, this work sets a foundation for a straightforward approach in measuring real‐time ketogenesis even as part of a routine MRI exam. This method, in particular, could serve as a powerful diagnostic tool for NAFLD and its transition to NASH.Support or Funding InformationNIH R01DK105346Sum spectra obtained by adding individual transients of the pseudo‐2D NMR acquisition post HP DHA injection. DHA and DHA‐hydrate resonances are not shown. Note dramatically increased BHB intensity in fasting. In the fasted state, 3‐ carbon precursors of glucose (alanine and lactate) show much reduced intensity.Figure 1

Citrate NMR peak irreproducibility in blood samples after reacquisition of spectra.

In our metabolomics studies we have noticed that repeated NMR acquisition on the same sample can result in altered metabolite signal intensities. To investigate the reproducibility of repeated NMR acquisition on selected metabolites in serum and plasma from two large human metabolomics studies. Two peak regions for each metabolite were integrated and changes occurring after reacquisition were correlated. Integral changes were generally small, but serum citrate signals decreased significantly in some samples. Several metabolite integrals were not reproducible in some of the repeated spectra. Following established protocols, randomising analysis order and biomarker validation are important.

Determination of hydroxyl groups in biorefinery resources via quantitative 31P NMR spectroscopy.

The analysis of chemical structural characteristics of biorefinery product streams (such as lignin and tannin) has advanced substantially over the past decade, with traditional wet-chemical techniques being replaced or supplemented by NMR methodologies. Quantitative 31P NMR spectroscopy is a promising technique for the analysis of hydroxyl groups because of its unique characterization capability and broad potential applicability across the biorefinery research community. This protocol describes procedures for (i) the preparation/solubilization of lignin and tannin, (ii) the phosphitylation of their hydroxyl groups, (iii) NMR acquisition details, and (iv) the ensuing data analyses and means to precisely calculate the content of the different types of hydroxyl groups. Compared with traditional wet-chemical techniques, the technique of quantitative 31P NMR spectroscopy offers unique advantages in measuring hydroxyl groups in a single spectrum with high signal resolution. The method provides complete quantitative information about the hydroxyl groups with small amounts of sample (~30 mg) within a relatively short experimental time (~30-120 min).

Interleaved Dual NMR Acquisition of Equivalent Transfer Pathways in TOCSY and HSQC Experiments.

A dual NMR data acquisition strategy to handle and detect two active equivalent transfer pathways is presented and discussed. We illustrate the power of this time-efficient approach by collecting two different 2D spectra simultaneously in a single experiment: i) TOCSY or HSQC-TOCSY spectra with different mixing times, ii) F2-13 C-coupled and decoupled HSQC spectra, iii) conventional and pure-shift HSQC spectra, or iv) complementary HSQC and HSQC-TOCSY spectra.

Rapid elucidation of chemical shift correlations in complex NMR spectra of organic molecules: Two-dimensional Hadamard pure shift NMR spectroscopy

Novel two dimensional Hadamard encoding/decoding based pure shift NMR acquisition techniques (TOCSY and HSQC) have been developed, which provide chemical shift information at ultra high resolution in very short spectrometer times. The efficacy of these methods for rapid assignment of chemical shifts in complex NMR spectra of organic molecules/natural products has been demonstrated. This would be of great help for rapid analysis of samples during separation of complex mixtures.

NMR metabolomic study of blood plasma in ischemic and ischemically preconditioned rats: an increased level of ketone bodies and decreased content of glycolytic products 24h after global cerebral ischemia.

Cardiac arrest is one of the leading causes of death among adults in older age. Understanding mechanisms how organism responds to ischemia at global level is essential for the prevention and ischemic patient's treatment. In this study, we used a global cerebral ischemia induced by four-vessel occlusion as an established animal model for ischemic stroke to investigate metabolic changes after 24h reperfusion, when transitions occur due to the onset of delayed neuronal death. We also focused on the endogenous phenomenon known as ischemic tolerance by the pre-ischemic treatment. The experiments were carried out on blood plasma samples as easily available and metabolically reflecting the overall changes in injured organism. Our results imply that disturbed glycolysis pathway, as a consequence of ischemic injury, leads to the increased level of ketone bodies (acetone, acetoacetate and β-hydroxybutyrate) along with increased utilization of triacylglycerols in plasma of ischemic and ischemically preconditioned rats. Complementary to, a decreased level of glycolytic intermediates (lactate, pyruvate, acetate) with increased level of glucose was found in ischemic and preconditioned animals. The protective effect of ischemic preconditioning on metabolome recovery was demonstrated by significantly increased level of creatine compared to ischemic, non-preconditioned rats. We also document that acetoacetate, pyruvate, lactate, and leucine have the best discriminatory power between ischemic and control plasma. Conclusively, our results provide evidence that NMR spectra analysis can identify specific group of metabolites present in plasma with the capability for discrimination between individual groups of animals. In addition, an excellent feasibility for the statistical discrimination among ischemic, preconditioned, and control rats can be applied regardless of native or deproteinated plasma and also regardless of noesy or cpmg NMR acquisition.