High-Resolution Two-Dimensional J-Resolved NMR Spectroscopy for Biological Systems

NMR spectroscopy is a principal tool in metabolomic studies and can, in theory, yield atom-level information critical for understanding biological systems. Nevertheless, NMR investigations on biological tissues generally have to contend with field inhomogeneities originating from variations in macroscopic magnetic susceptibility; these field inhomogeneities broaden spectral lines and thereby obscure metabolite signals. The congestion in one-dimensional NMR spectra of biological tissues often leads to ambiguities in metabolite identification and quantification. We propose an NMR approach based on intermolecular double-quantum coherences to recover high-resolution two-dimensional (2D) J-resolved spectra from inhomogeneous magnetic fields, such as those created by susceptibility variations in intact biological tissues. The proposed method makes it possible to acquire high-resolution 2D J-resolved spectra on intact biological samples without recourse to time-consuming shimming procedures or the use of specialized hardware, such as magic-angle-spinning probes. Separation of chemical shifts and J couplings along two distinct dimensions is achieved, which reduces spectral crowding and increases metabolite specificity. Moreover, the apparent J coupling constants observed are magnified by a factor of 3, facilitating the accurate measurement of small J couplings, which is useful in metabolic analyses. Dramatically improved spectral resolution is demonstrated in our applications of the technique on pig brain tissues. The resulting spectra contain a wealth of chemical shift and J-coupling information that is invaluable for metabolite analyses. A spatially localized experiment applied on an intact fish (Crossocheilus siamensis) reveals the promise of the proposed method in in vivo metabolite studies. Moreover, the proposed method makes few demands on spectrometer hardware and therefore constitutes a convenient and effective manner for metabonomics study of biological systems.     

An Introduction to Biological NMR Spectroscopy

NMR spectroscopy is a powerful tool for biologists interested in the structure, dynamics, and interactions of biological macromolecules. This review aims at presenting in an accessible manner the requirements and limitations of this technique. As an introduction, the history of NMR will highlight how the method evolved from physics to chemistry and finally to biology over several decades. We then introduce the NMR spectral parameters used in structural biology, namely the chemical shift, the J-coupling, nuclear Overhauser effects, and residual dipolar couplings. Resonance assignment, the required step for any further NMR study, bears a resemblance to jigsaw puzzle strategy. The NMR spectral parameters are then converted into angle and distances and used as input using restrained molecular dynamics to compute a bundle of structures. When interpreting a NMR-derived structure, the biologist has to judge its quality on the basis of the statistics provided. When the 3D structure is a priori known by other means, the molecular interaction with a partner can be mapped by NMR: information on the binding interface as well as on kinetic and thermodynamic constants can be gathered. NMR is suitable to monitor, over a wide range of frequencies, protein fluctuations that play a crucial role in their biological function. In the last section of this review, intrinsically disordered proteins, which have escaped the attention of classical structural biology, are discussed in the perspective of NMR, one of the rare available techniques able to describe structural ensembles. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 16 MCP).     

A Study of Water–Protein Interactions by High-Resolution NMR Spectroscopy

The interaction between carbonic anhydrase B in the molten globule state and water molecules was studied by high-resolution NMR spectroscopy. NMR spin diffusion experiments revealed spin diffusion propagation from the protein to waters. This is a process of complex bioexponential kinetics presented in spin diffusion spectra as a change in water signal intensity dependent on the protein postexcitation time. Its reverse, spin diffusion propagation from waters to the protein, was also found. These phenomena are protein concentration- and temperature-dependent and shown to be possibly explained with the assumption that there exist water–protein complexes provoking formation of large branched associations. At a temperature above 309 K, a stepwise increase in the interaction between water and proteins occurs in these complexes. The formation of water–protein associations is induced by increasing temperature and/or protein concentration. In these associations, at normal temperature, the protein mobility is close to that of carbonic anhydrase B dimers.     

High-Resolution Two-Dimensional Atomic Localization Via Tunable Surface Plasmon Polaritons

The two-dimensional (2D) atomic localization is theoretically investigated via tunable surface plasmon polaritons (SPPs), generated on the metal (Ag) surface coupled to a quantum coherent three-level \(\lambda\)-type medium (\(^{87}\)Rb) embedded as a dielectric host. Such a useful scheme for highly precise atomic localization is reported by using the absorption spectrum of SPPs. Owing to space-dependent light–matter interaction, the sharp localized peaks are observed in a single wavelength domain of 2D space with maximum probability. By properly varying the system parameters, the precision and numbers of the localized peaks are controlled. Consequently, highly efficient and high-resolution atomic localization can be achieved in a region smaller than \(\lambda /20\times \lambda /20\). The spatial resolution of atomic localization is greatly improved as compared to the previously studied cases. These results may have potential useful applications in the fields of quantum nanoplasmonics, nanolithography, and nanophotonics.

     

Discrimination of Basal Cell Carcinoma from Normal Skin Tissue Using High-Resolution Magic Angle Spinning 1H NMR Spectroscopy

High-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy is a useful tool for investigating the metabolism of various cancers. Basal cell carcinoma (BCC) is the most common skin cancer. However, to our knowledge, data on metabolic profiling of BCC have not been reported in the literature. The objective of the present study was to investigate the metabolic profiling of cutaneous BCC using HR-MAS ¹H NMR spectroscopy. HR-MAS ¹H NMR spectroscopy was used to analyze the metabolite profile and metabolite intensity of histopathologically confirmed BCC tissues and normal skin tissue (NST) samples. The metabolic intensity normalized to the total spectral intensities in BCC and NST was compared, and multivariate analysis was performed with orthogonal partial least-squares discriminant analysis (OPLS-DA). P values < 0.05 were considered statistically significant. Univariate analysis revealed 9 metabolites that showed statistically significant difference between BCC and NST. In multivariate analysis, the OPLS-DA models built with the HR-MAS NMR metabolic profiles revealed a clear separation of BCC from NST. The receiver operating characteristic curve generated from the results revealed an excellent discrimination of BCC from NST with an area under the curve (AUC) value of 0.961. The present study demonstrated that the metabolite profile and metabolite intensity differ between BCC and NST, and that HR-MAS ¹H NMR spectroscopy can be a valuable tool in the diagnosis of BCC.     

High-Resolution Proton NMR Studies of Lymphocyte Extracts

Anatomic imaging is now a well-developed application of magnetic resonance. Greater capabilities for physiologic characterization should become possible by concomitant application of spectroscopic methods. High-resolution in vitro spectroscopy must first provide a framework upon which in vivo and diagnostic interpretation may be based. Biochemical profiles consisting of quantitation of extracted aqueous metabolites and lipids of particular cells or organs establish an in vitro glossary for what may be found in the intact cell or living subject. A large variety of amino acids, intermediary metabolites, membrane precursors, and nucleotides are detectable in extracts of human peripheral blood lymphocytes, and significant changes in intracellular concentrations have been monitored after lectin-induced activation. Corresponding changes in lipid profile have also been noted. An increasing variety of other cells and tissues are being similarly characterized. Despite its limitations, NMR analysis possesses the unique prospect of providing a noninvasive and nondestructive source of biochemical information.     

A Novel Detection Scheme for High-Resolution Two-Dimensional Spin-Echo Correlated Spectra in Inhomogeneous Fields

Background

Two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy is a powerful and non-invasive tool for the analysis of molecular structures, conformations, and dynamics. However, the inhomogeneity of magnetic fields experienced by samples will destroy spectral information and hinder spectral analysis. In this study, a new pulse sequence is proposed based on the modulation of distant dipolar field to recover high-resolution 2D spin-echo correlated spectroscopy (SECSY) from inhomogeneous fields.

Method and Material

By using the new sequence, the correlation information between coupled spins and the J coupled information with straightforward multiplet patterns can be obtained free from inhomogeneous line broadening. In addition, the new sequence is also suitable for non-J coupled spin systems. Although three-dimensional acquisition is needed, the evolution of indirect detection dimensions is carefully designed and the ultrafast acquisition scheme is utilized to improve the acquisition efficiency. A chemical solution of butyl methacrylate (C₈H₁₄O₂) in DMSO (C₂H₆SO) in a deshimmed magnetic field was tested to demonstrate the implementation details of the new sequence. The performance of the new sequence relative to the conventional SECSY sequence was shown by using an aqueous solution of main brain metabolites in a deshimmed magnetic field.

Conclusion

The results reveal that the new sequence provides an attractive way to eliminate the inhomogeneous spectral line broadening for the spin-echo correlated spectrum and is a promising tool for the study of metabolites in metabonomics, even for the applications on in vivo and in situ high-resolution 2D NMR spectroscopy.     

High-Resolution 1H NMR Spectroscopy of Fish Muscle,Eggs and Small Whole Fish via Hadamard-Encoded Intermolecular Multiple-Quantum Coherence

Background and Purpose

Nuclear magnetic resonance (NMR) spectroscopy has become an important technique for tissue studies. Since tissues are in semisolid-state, their high-resolution (HR) spectra cannot be obtained by conventional NMR spectroscopy. Because of this restriction, extraction and high-resolution magic angle spinning (HR MAS) are widely applied for HR NMR spectra of tissues. However, both of the methods are subject to limitations. In this study, the feasibility of HR ¹H NMR spectroscopy based on intermolecular multiple-quantum coherence (iMQC) technique is explored using fish muscle, fish eggs, and a whole fish as examples.

Materials and Methods

Intact salmon muscle tissues, intact eggs from shishamo smelt and a whole fish (Siamese algae eater) are studied by using conventional 1D one-pulse sequence, Hadamard-encoded iMQC sequence, and HR MAS.

Results

When we use the conventional 1D one-pulse sequence, hardly any useful spectral information can be obtained due to the severe field inhomogeneity. By contrast, HR NMR spectra can be obtained in a short period of time by using the Hadamard-encoded iMQC method without shimming. Most signals from fatty acids and small metabolites can be observed. Compared to HR MAS, the iMQC method is non-invasive, but the resolution and the sensitivity of resulting spectra are not as high as those of HR MAS spectra.

Conclusion

Due to the immunity to field inhomogeneity, the iMQC technique can be a proper supplement to HR MAS, and it provides an alternative for the investigation in cases with field distortions and with samples unsuitable for spinning. The acquisition time of the proposed method is greatly reduced by introduction of the Hadamard-encoded technique, in comparison with that of conventional iMQC method.     

Functional Groups Determine Biochar Properties (pH and EC) as Studied by Two-Dimensional 13C NMR Correlation Spectroscopy

While the properties of biochar are closely related to its functional groups, it is unclear under what conditions biochar develops its properties. In this study, two-dimensional (2D) ¹³C nuclear magnetic resonance (NMR) correlation spectroscopy was for the first time applied to investigate the development of functional groups and establish their relationship with biochar properties. The results showed that the agricultural biomass carbonized to biochars was a dehydroxylation/dehydrogenation and aromatization process, mainly involving the cleavage of O-alkylated carbons and anomeric O-C-O carbons in addition to the production of fused-ring aromatic structures and aromatic C-O groups. With increasing charring temperature, the mass cleavage of O-alkylated groups and anomeric O-C-O carbons occurred prior to the production of fused-ring aromatic structures. The regression analysis between functional groups and biochar properties (pH and electrical conductivity) further demonstrated that the pH and electrical conductivity of rice straw derived biochars were mainly determined by fused-ring aromatic structures and anomeric O-C-O carbons, but the pH of rice bran derived biochars was determined by both fused-ring aromatic structures and aliphatic O-alkylated (HCOH) carbons. In summary, this work suggests a novel tool for characterising the development of functional groups in biochars.     

Muon Spectroscopy Applied to Biological Systems: A Study of Thiyl Radicals, RS

Thiyl radicals (RS.) are formed when positive muons (μ⁺) are implanted into solutions of thiocarbonyl compounds (C = S) in ethanol, tetrahydrofuran or formamide solvents. A solvent dependence is found, which reflects changing electronic interactions and overall conformations, suggesting that the properties of RS. radicals may be dependent on the polarity (philicity) of the environment in which they are formed.     

生物源性基因疫苗的运送系统

基因疫苗具有很多独特的优点,已经成为疫苗研究领域的热点。但由于其免疫原性相对较弱,限制了基因疫苗的广泛应用。人们一直在寻求一种理想的基因疫苗运送系统,它不仅能将基因疫苗导入体内,还能提高基因疫苗的免疫原性,诱导机体产生持续高水平的免疫应答反应。     

Determination of Reference Ultrasound Parameters for Model and Hydrofluoroalkane Propellants Using High-Resolution Ultrasonic Spectroscopy

The aim of this research was to determine the reference ultrasonic velocity (v) and attenuation coefficient (α) for 2H, 3H-perfluoropentane (HPFP), 1,1,1,2-tetrafluoroethane (HFA-134a) and 1,1,1,2,3,3,3-tetrafluoroethane (HFA-227) propellants, for the future purpose of characterising pressurised metered dose inhaler (pMDI) formulations using high-resolution ultrasonic spectroscopy (HRUS). Perfluoroheptane (PFH) was used as a reference material for HPFP. With its velocity and attenuation coefficient determined at 25 °C, HPFP was subsequently used as a reference for HFA-134a and HFA-227. It was found that there is a linear decline in ultrasonic velocity with an increase in temperature. As with HPFP, the ultrasonic velocity of HFA-134a and HFA-227 were successfully calculated at 25 °C. However, the difference in density and viscosity between reference and sample prevented accurate determination of reference attenuation coefficient for the hydrofluoroalkanes. With ultrasonic velocity alone, dispersion concentration and stability monitoring for experimental pMDI formulations is possible using HRUS. However, at this point in time measurement of particle size is not feasible.     

Zwitterionic Phosphorylated Quinines as Chiral Solvating Agents for NMR Spectroscopy

Because of their unique 3D arrangement, naturally occurring Cinchona alkaloids and their synthetic derivatives have found wide‐ranging applications in chiral recognition. Recently, we determined the enantioselective properties of C‐9‐phosphate mixed triesters of quinine as versatile chiral solvating agents in nuclear magnetic resonance (NMR) spectroscopy. In the current study, we introduce new zwitterionic members of this class of molecules containing a negatively charged phosphate moiety (i.e., ethyl, n‐butyl and phenyl hydrogen quininyl phosphate). An efficient approach for synthesizing these compounds is elaborated, and full characterization, including conformational and autoaggregation phenomena studies, was performed. Therefore, their ability to induce NMR anisochrony of selected enantiomeric substrates (i.e., primarily N‐DNB‐protected amino acids and their methyl esters) was analyzed compared to uncharged diphenyl quininyl phosphate and its positively charged quaternary ammonium hydrochloride salt. In addition, ¹H and ¹³C NMR experiments revealed their enantiodiscrimination potential toward novel analytes, such as secondary amines and nonprotected amino acids. Chirality 27:752–760, 2015. © 2015 Wiley Periodicals, Inc.     

Biological 1H NMR spectroscopy

Proton nuclear magnetic resonance spectroscopy (1H NMR) is a powerful analytical method used to identify and quantitate chemical compounds. In recent years, it has been used to study rates of metabolism in microbes, isolated perfused tissues, intact animals, and human beings. This review highlights some of the more recent biological applications of 1H NMR in the study of metabolic pathophysiology in animals and man. 1H NMR can rapidly analyze complex mixtures of metabolites found in body fluid and biopsy specimens. In vivo 1H NMR methods can measure intracellular pH, a wide variety of metabolites, tissue perfusion, and rates of metabolism of endogenous and exogenous compounds. Using 13C labeled compounds or magnetization transfer techniques metabolic fluxes may be measured in vivo during virtually all normal and abnormal physiological conditions.     

Two-Dimensional Solid-State NMR Applied to a Chimeric Potassium Channel

Solid-state NMR (ssNMR) represents a spectroscopic method to study membrane protein structure and dynamics in lipid bilayers. We present two-dimensional correlation experiments conducted on a fully [¹³C,¹⁵N] labeled version of a chimeric potassium (KcsA-Kv1.3) channel. Data obtained by using two different ion concentrations suggest a structural conservation of the selectivity filter region. SsNMR experiments conducted at two different temperatures point to differential molecular dynamics of the channel.