SWET for Secure Water Suppression on Probes with High Quality Factor

Water suppression by selective preirradiation is increasingly difficult to achieve on probeheads with high quality factor because of the opposing forces of radiation damping. Here we show that a simple modification to the WET scheme provides reliable water suppression in aqueous solutions of proteins and peptides with minimal saturation of the H^α protons. The scheme is shown to work also with dilute peptide solutions. It is recommended to maintain the water suppression during the evolution time of COSY experiments by weak selective irradiation that causes only minimal Bloch-Siegert shifts. The new water-suppression scheme suppresses the water magnetization by spatial scrambling. Traditional water suppression by preirradiation is similarly based more on water scrambling due to the radiofrequency inhomogeneity than on relaxation effects.Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Metabolomic analysis using optimized NMR and statistical methods

NMR-based metabolomics requires robust automated methodologies, and the accuracy of NMR-based metabolomics data is greatly influenced by the reproducibility of data acquisition and processing methods. Effective water resonance signal suppression and reproducible spectral phasing and baseline traces across series of related samples are crucial for statistical analysis. We assess robustness, repeatability, sensitivity, selectivity, and practicality of commonly used solvent peak suppression methods in the NMR analysis of biofluids with respect to the automated processing of the NMR spectra and the impact of pulse sequence and data processing methods on the sensitivity of pattern recognition and statistical analysis of the metabolite profiles. We introduce two modifications to the excitation sculpting pulse sequence whereby the excitation solvent suppression pulse cascade is preceded by low-power water resonance presaturation pulses during the relaxation delay. Our analysis indicates that combining water presaturation with excitation sculpting water suppression delivers the most reproducible and information-rich NMR spectra of biofluids.     

Protein hydration studied with homonuclear 3D1H NMR experiments

Summary Homonuclear 3D¹H NOESY-TOCSY and 3D¹H ROESY-TOCSY experiments were used to resolve and assign nuclear Overhauser effect (NOE) cross peaks between the water signal and individual polypeptide proton resonances in H₂O solutions of the basic pancreatic trypsin inhibitor. Combined with a novel, robust water-suppression technique, positive and negative intermolecular NOEs were detected at 4°C. The observation of positive NOEs between water protons and protein protons enables more precise estimates of the very short residence times of the water molecules in the hydration sites on the protein surface.     

Profiling lipid metabolites yields unique information on sex- and time-dependent responses of fathead minnows (Pimephales promelas) exposed to 17α-ethynylestradiol

Alterations in hepatic lipid profiles of fathead minnows (FHM) exposed to the synthetic estrogen 17α-ethynylestradiol (EE2) were determined using ¹H-NMR spectroscopy-based metabolite profiling. The exposures were conducted using either 10 ng/l or 100 ng/l EE2 via a continuous flow water delivery system. Livers were collected at 1, 4, and 8 days of the exposure and 8 days after the chemical was removed from the water (i.e. an 8 day depuration). The exposure resulted in a number of sex-specific changes in lipid profiles that were also highly time dependent. Those metabolites most affected by exposure included phosphatidylcholine, diglycerides, triglycerides and cholesterol. In addition, changes in the length and degree of unsaturation of hepatic fatty acids were observed. Lipid profiles in plasma for fish collected on the 4th day of exposure were also analyzed in order to provide further insights into changes observed in hepatic metabolite changes. Using validated partial least-squares discriminant analysis (PLS-DA), the response trajectories of the male liver lipid profiles at both exposure concentrations were compared. This analysis indicated that the males exposed to the low concentration of EE2 (10 ng/l) were largely able to recover from the exposure once the chemical was removed from the water. Conversely, the males exposed to the high concentration (100 ng/l) did not appear to recover from the exposure despite the 8 day depuration.     

Variation of metabolites in normal human urine

Urine is often sampled from patients participating in clinical and metabolomic studies. Biological homeostasis occurs in humans, but little is known about the variability of metabolites found in urine. It is important to define the inter- and intra-individual metabolite variance within a normal population before scientific or clinical conclusions are made regarding different pathophysiologies. This study investigates the variability of selected urine metabolites in a group of 60 healthy men and women over a period of 30 days. To monitor individual variation, 6 women from the normal population were randomly selected and followed for 30 days. To determine the influence of extraneous environmental factors urine was collected from 25 guinea pigs with similar genetics, diet, and living environment. For both studies, 24 metabolites were identified and quantified using high-resolution ¹H nuclear magnetic resonance spectroscopy (NMR). The data demonstrated large inter and intra-individual variation in metabolite concentrations in both normal human and control animal populations. A defined normal baseline is essential before any conclusions may be drawn regarding changes in urine metabolite concentrations.     

BioMagResBank (BMRB) as a partner in the Worldwide Protein Data Bank (wwPDB): new policies affecting biomolecular NMR depositions

We describe the role of the BioMagResBank (BMRB) within the Worldwide Protein Data Bank (wwPDB) and recent policies affecting the deposition of biomolecular NMR data. All PDB depositions of structures based on NMR data must now be accompanied by experimental restraints. A scheme has been devised that allows depositors to specify a representative structure and to define residues within that structure found experimentally to be largely unstructured. The BMRB now accepts coordinate sets representing three-dimensional structural models based on experimental NMR data of molecules of biological interest that fall outside the guidelines of the Protein Data Bank (i.e., the molecule is a peptide with 23 or fewer residues, a polynucleotide with 3 or fewer residues, a polysaccharide with 3 or fewer sugar residues, or a natural product), provided that the coordinates are accompanied by representation of the covalent structure of the molecule (atom connectivity), assigned NMR chemical shifts, and the structural restraints used in generating model. The BMRB now contains an archive of NMR data for metabolites and other small molecules found in biological systems.     

Watergate技术在DNA上的应用

比较了Watergate和其他几种不同的压水峰方法,结果表明Watergate是效果较好,实验上容易实现的水峰抑制方法.用Watergate-NOESY及Watergate-TOCSY,完成了转录因子E2F所识别的DNA双链片段d(5′-TTTCGCGC)·d(3′-AAAGCGCG)中大部分G,C碱基上可交换质子的归属.     

Use of EDTA to minimize ionic strength dependent frequency shifts in the <Superscript>1</Superscript>H NMR spectra of urine

The ¹H NMR spectrum of urine exhibits a large number of detectable metabolites and is, therefore, highly suitable for the study of perturbations caused by disease, toxicity, nutrition or environmental factors in humans and animals. However, variations in the chemical shifts and intensities due to altered pH and ionic strength present a challenge in NMR-based studies. With a view towards understanding and minimizing the effects of these variations, we have extensively studied the effects of ionic strength and pH on the chemical shifts of common urine metabolites and their possible reduction using EDTA (ethylenediaminetetraacetic acid). ¹H NMR chemical shifts for alanine, citrate, creatinine, dimethylamine, glycine, histidine, hippurate, formate and the internal reference, TSP (trimethylsilylpropionic acid-d₄, sodium salt) obtained under different conditions were used to assess each effect individually. EDTA minimizes the frequency shifts of the metabolites that have a propensity for metal binding. Chelation of such metal ions is evident from the appearance of signals from EDTA complexed to divalent metal ions such as calcium and magnesium. Not surprisingly, increasing the buffer concentration or buffer volume also minimizes pH dependent frequency shifts. The combination of EDTA and an appropriate buffer effectively minimizes both pH dependent frequency shifts and ionic strength dependent intensity variations in urine NMR spectra. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Use of a water flip-back pulse in the homonuclear NOESY experiment

Summary A simple modification to the WATERGATE water suppression scheme [Piotto, M., Saudek, V. and Sklená, V. (1992) J. Biomol. NMR, 2, 661–665] is proposed. Radiation damping is used as an active element during the mixing time of a NOESY experiment, in order to obtain a reproducable state of the water magnetization at the end of the mixing time. Through the use of a water flip-back pulse and a gradient-tailored excitation scheme, we obtain both an excellent water suppression and a water magnetization close to equilibrium at the beginning of the acquisition time. We show experimentally that this modification results in a 20% gain in intensity for all signals when using a relaxation delay of 1.5 s, and also that avoiding a semisaturated state for the water magnetization allows the amide protons as well as other proton resonances to relax to equilibrium with their proper relaxation time.     

Validation of a urine metabolome fingerprint in dog for phenotypic classification

Selective breeding of dogs over hundreds of years has inadvertently resulted in breed-specific propensities to particular diseases. Furthermore, it has likely induced more subtle affects on the physiology of certain breeds and moved them from their evolutionary optima. In the absence of obvious disease phenotypes such subtle changes could have yet unrecognised breed-specific implications for health and well-being. Here we have applied NMR metabolomics as a discovery-driven approach to identify the impact of breed on the urinary profile of dog and to determine if non-disease-related breed differences can be identified. Multiple urines were collected non-invasively over a two-week period from seven neutered male Labrador retrievers and miniature Schnauzers. Following NMR analyses by 1-dimensional ¹H and 2-dimensional ¹H J-resolved (JRES) spectroscopy, principal component analysis revealed that the metabolic variability within each individual is relatively small compared to inter-individual variability, and that some separation between breeds was evident. A supervised model, using partial least squares discriminant analysis (PLS-DA) with class based upon breed, was trained using the JRES data. The model predicted correctly the breed of seven additional urines, yielding a model sensitivity and specificity of 100%. Several significant metabolic differences between the breeds were identified. A second model was developed using PLS-DA with class based upon individual dogs, which again achieved high classification accuracy for the test set. Overall, this confirms that canine urine is information-rich and that breed is a major determinant of urinary metabolic fingerprints. In the future this may enable a more accurate development of specific nutritional care for an individual or breed.     

Understanding the role of hydrogen bonds in water dynamics and protein stability

The mechanisms of cold and pressure denaturation of proteins are a matter of debate, but it is commonly accepted that water plays a fundamental role in the process. It has been proposed that the denaturation process is related to an increase of hydrogen bonds among hydration water molecules. Other theories suggest that the causes of denaturation are the density fluctuations of surface water, or the destabilization of hydrophobic contacts as a consequence of water molecule inclusions inside the protein, especially at high pressures. We review some theories that have been proposed to give insight into this problem, and we describe a coarse-grained model of water that compares well with experiments for proteins’ hydration water. We introduce its extension for a homopolymer in contact with the water monolayer and study it by Monte Carlo simulations in an attempt to understand how the interplay of water cooperativity and interfacial hydrogen bonds affects protein stability.     

The impact of free or standardized lifestyle and urine sampling protocol on metabolome recognition accuracy

Urine contains a clear individual metabolic signature, although embedded within a large daily variability. Given the potential of metabolomics to monitor disease onset from deviations from the “healthy” metabolic state, we have evaluated the effectiveness of a standardized lifestyle in reducing the “metabolic” noise. Urine was collected from 24 (5 men and 19 women) healthy volunteers over a period of 10 days: phase I, days 1–7 in a real-life situation; phase II, days 8–10 in a standardized diet and day 10 plus exercise program. Data on dietary intake and physical activity have been analyzed by a nation-specific software and monitored by published protocols. Urine samples have been analyzed by ¹H NMR followed by multivariate statistics. The individual fingerprint emerged and consolidated with increasing the number of samples and reaches ~100 % cross-validated accuracy for about 40 samples. Diet standardization reduced both the intra-individual and the interindividual variability; the effect was due to a reduction in the dispersion of the concentration values of several metabolites. Under standardized diet, however, the individual phenotype was still clearly visible, indicating that the individual’s signature was a strong feature of the metabolome. Consequently, cohort studies designed to investigate the relation of individual metabolic traits and nutrition require multiple samples from each participant even under highly standardized lifestyle conditions in order to exploit the analytical potential of metabolomics. We have established criteria to facilitate design of urine metabolomic studies aimed at monitoring the effects of drugs, lifestyle, dietary supplements, and for accurate determination of signatures of diseases.