Extended automated quantification algorithm (AQuA) for targeted 1H NMR metabolomics of highly complex samples: application to plant root exudates.

INTRODUCTION: The Automated Quantification Algorithm (AQuA) is a rapid and efficient method for targeted NMR-based metabolomics, currently optimised for blood plasma. AQuA quantifies metabolites from 1D-1H NMR spectra based on the height of only one signal per metabolite, which minimises the computational time and workload of the method without compromising the quantification accuracy. OBJECTIVES: To develop a fast and computationally efficient extension of AQuA for quantification of selected metabolites in highly complex samples, with minimal prior sample preparation. In particular, the method should be capable of handling interferences caused by broad background signals. METHODS: An automatic baseline correction function was combined with AQuA into an automated workflow, the extended AQuA, for quantification of metabolites in plant root exudate NMR spectra that contained broad background signals and baseline distortions. The approach was evaluated using simulations as well as a spike-in experiment in which known metabolite amounts were added to a complex sample matrix. RESULTS: The extended AQuA enables accurate quantification of metabolites in 1D-1H NMR spectra with varying complexity. The method is very fast (< 1 s per spectrum) and can be fully automated. CONCLUSIONS: The extended AQuA is an automated quantification method intended for 1D-1H NMR spectra containing broad background signals and baseline distortions. Although the method was developed for plant root exudates, it should be readily applicable to any NMR spectra displaying similar issues as it is purely computational and applied to NMR spectra post-acquisition.

Determination of Amide cis/trans Isomers in N-Acetyl-d-glucosamine: Tailored NMR Analysis of the N-Acetyl Group Conformation.

N-Acetyl-d-glucosamine (GlcNAc) is one of the most common amino sugars in nature, but the conformation of its N-acetyl group has drawn little attention. We report herein the first identification of NH protons of the amide cis forms of α- and ß-GlcNAc by NMR spectroscopy. Relative quantification and thermodynamic analysis of both cis and trans forms was carried out in aqueous solution. The NH protons were further utilized by adapting protein NMR experiments to measure eight J-couplings within the N-acetyl group, of which six are sensitive to the H2-NH conformation and two are sensitive to the amide conformation. For amide cis and trans forms, the orientation between H2 and NH was determined as anti conformation, while a small percentage of syn conformation was predicted for the amide trans form of ß-GlcNAc. This approach holds great promise for the detailed conformational analysis of GlcNAc in larger biomolecules, such as glycoproteins and polysaccharides.

A detailed picture of a protein-carbohydrate hydrogen-bonding network revealed by NMR and MD simulations.

Cyanovirin-N (CV-N) is a cyanobacterial lectin with antiviral activity towards HIV and several other viruses. Here, we identify mannoside hydroxyl protons that are hydrogen bonded to the protein backbone of the CV-N domain B binding site, using NMR spectroscopy. For the two carbohydrate ligands Manα(1→2)ManαOMe and Manα(1→2) Manα(1→6)ManαOMe five hydroxyl protons are involved in hydrogen-bonding networks. Comparison with previous crystallographic results revealed that four of these hydroxyl protons donate hydrogen bonds to protein backbone carbonyl oxygens in solution and in the crystal. Hydrogen bonds were not detected between the side chains of Glu41 and Arg76 with sugar hydroxyls, as previously proposed for CV-N binding of mannosides. Molecular dynamics simulations of the CV-N/Manα(1→2)Manα(1→6)ManαOMe complex confirmed the NMR-determined hydrogen-bonding network. Detailed characterization of CV-N/mannoside complexes provides a better understanding of lectin-carbohydrate interactions and opens up to the use of CV-N and similar lectins as antiviral agents.

Modulating Surface Properties of the Linothele fallax Spider Web by Solvent Treatment.

Linothele fallax (Mello-Leitão) (L. fallax) spider web, a potentially attractive tissue engineering material, was investigated using quantitative peak force measurement atomic force microscopy and scanning electron microscopy with energy dispersive spectroscopy both in its natural state and after treatment with solvents of different protein affinities, namely, water, ethanol, and dimethyl sulfoxide (DMSO). Native L. fallax silk threads are densely covered by globular objects, which constitute their inseparable parts. Depending on the solvent, treating L. fallax modifies its appearance. In the case of water and ethanol, the changes are minor. In contrast, DMSO practically removes the globules and fuses the threads into dense bands. Moreover, the solvent treatment influences the chemistry of the threads' surface, changing their adhesive and, therefore, biocompatibility and cell adhesion properties. On the other hand, the solvent-treated web materials' contact effect on different types of biological matter differs considerably. Protein-rich matter controls humidity better when wrapped in spider silk treated with more hydrophobic solvents. However, carbohydrate plant materials retain more moisture when wrapped in native spider silk. The extracts produced with the solvents were analyzed using nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry techniques, revealing unsaturated fatty acids as representative adsorbed species, which may explain the mild antibacterial effect of the spider silk. The extracted metabolites were similar for the different solvents, meaning that the globules were not "dissolved" but "fused into" the threads themselves, being supposedly rolled-in knots of the protein chain.

Direct Observation of Carbohydrate Hydroxyl Protons in Hydrogen Bonds with a Protein.

Hydroxyl proton resonances of uniformly 13C-labeled Manα(1-2)Manα(1-2)ManαOMe (Man3) bound to cyanovirin-N (CV-N) were detected at ambient temperature in aqueous solution by NMR spectroscopy. The directions of the hydroxyl groups were determined on the basis of NOEs, and a previously unknown hydrogen-bonding network between Man3 and CV-N was discovered. This is the first report on detecting hydroxyl protons of a protein-bound carbohydrate in aqueous solution by NMR. Approaches such as those presented here may open the door for accurately determining intermolecular hydrogen bonds in carbohydrate-protein complexes.

Exploiting Uniformly 13C-Labeled Carbohydrates for Probing Carbohydrate-Protein Interactions by NMR Spectroscopy.

NMR of a uniformly 13C-labeled carbohydrate was used to elucidate the atomic details of a sugar-protein complex. The structure of the 13C-labeled Manα(1-2)Manα(1-2)ManαOMe trisaccharide ligand, when bound to cyanovirin-N (CV-N), was characterized and revealed that in the complex the glycosidic linkage torsion angles between the two reducing-end mannoses are different from the free trisaccharide. Distances within the carbohydrate were employed for conformational analysis, and NOE-based distance mapping between sugar and protein revealed that Manα(1-2)Manα(1-2)ManαOMe is bound more intimately with its two reducing-end mannoses into the domain A binding site of CV-N than with the nonreducing end unit. Taking advantage of the 13C spectral dispersion of 13C-labeled carbohydrates in isotope-filtered experiments is a versatile means for a simultaneous mapping of the binding interactions on both, the carbohydrate and the protein.

1H, 13C and 15N backbone resonance assignment of Cel45A from Phanerochaete chrysosporium.

A glycoside hydrolase family 45 (GH45) enzyme from the white-rot basidiomycete fungus Phanerochaete chrysosporium (PcCel45A) was expressed in Pichia pastoris with 13C and 15N labelling. A nearly complete assignment of 1H, 13C and 15N backbone resonances was obtained, as well as the secondary structure prediction based on the assigned chemical shifts using the TALOS-N software. The predicted secondary structure was almost identical to previously published crystal structures of the same enzyme, except for differences in the termini of the sequence. This is the first NMR study using an isotopically labelled GH45 enzyme.

Impact of mutations in starch synthesis genes on morphological, compositional, molecular structure, and functional properties of potato starch.

Morphology, composition and molecular structure of starch directly affect the functional properties. This study investigated the morphological, compositional, and molecular structure properties of starch from starch branching enzyme gene (SBE) and granule-bound starch synthase gene (GBSS) mutated potato, and their associations with thermal, pasting, and film-making properties. SBE mutations were induced in native variety Desiree while GBSS mutations were herestacked to a selected SBE mutated parental line. Mutations in SBE resulted in smaller starch granules and higher amylose content, while GBSS mutations in the SBE background reduced amylose content. Mutations in SBE, particularly with GBSS mutations, significantly increased total phosphorus content. 31P NMR spectroscopy revealed higher proportions of C6-bound phosphate than of C3-bound phosphate in all studied lines. Amylopectin unit chain and internal chain distributions showed higher proportions of long chains in mutated lines compared with Desiree. These amylopectin long-chains were positively correlated with gelatinizationand, pasting temperatures, and temperature at peak viscosity. Short amylopectin chains showed positive correlations with breakdown viscosity, but negative correlations with the crystal melting temperature of retrograded starch. Total phosphorus content was positively correlated with the crystal melting temperature of retrograded starch. Starch from different lines was used to produce a series of potato starch films that differed in morphology and functional properties. A negative correlation was observed between Young's modulus of films and the long amylopectin-chain fraction. Thermal gravimetric analysis revealed highest thermal stability of Desiree starch films, followed by films from SBE-mutated high-amylose lines. Oxygen transmission rate and oxygen permeability analyses showed that films made with starch from selected GBSS and SBEs mutated line maintained comparable oxygen barrier properties to Desiree film. These insights on the impact of genetic mutations on starch properties indicate potential applications of in-planta starch modification for specific end-uses including packaging.

Metabolic labeling of hyaluronan: Biosynthesis and quantitative analysis of 13C,15N-enriched hyaluronan by NMR and MS-based methods.

Hyaluronan (HA), a member of the GAG family of glycans, has many diverse biological functions that vary a lot depending on the length of the HA chain and its concentration. A better understanding of the structure of different-sized HA at the atomic level is therefore crucial to decipher these biological functions. NMR is a method of choice for conformational studies of biomolecules, but there are limitations due to the low natural abundance of the NMR active nuclei 13C and 15N. We describe here the metabolic labeling of HA using the bacterium Streptococcus equi subsp. Zooepidemicus and the subsequent analysis by NMR and mass spectrometry. The level of 13C and 15N isotope enrichment at each position was determined quantitatively by NMR spectroscopy and was further confirmed by high-resolution mass spectrometry analysis. This study provides a valid methodological approach that can be applied to the quantitative assessment of isotopically labeled glycans and will help improve detection capabilities and facilitate future structure-function relationship analysis of complex glycans.

Complete 1H and 13C NMR spectral assignment of d-glucofuranose.

Although d-glucose is the most common sugar in nature, only a few NMR studies have focused on its minor furanose forms, and they have been limited to the anomeric position. Here, complete 1H and 13C NMR spectral analysis of α- and ß-d-glucofuranose was performed, including signal assignment, chemical shifts, and coupling constants. Selective and non-selective 1D and 2D NMR experiments were used for the analysis, complemented by spin simulations and iterative spectral analysis.

Factors influencing stoichiometry and stability of polyoxometalate - peptide complexes.

In the pursuit of understanding the factors guiding interactions between polyoxometalates (POMs) and biomolecules, several complexes between Keggin phosphomolybdate and diglycine have been produced at different acidity and salinity conditions, leading to difference in stoichiometry and in crystal structure. Principal factors determining how the POM and dipeptide interact appear to be pH, ionic strength of the medium, and the molar ratio of POM to peptide. An important effect turned out to be even the structure-directing role of the sodium cations coordinating carbonyl functions of the peptide bond. Given the interest in applying POMs in biological systems, these factors are highly relevant to consider. In the view of recent interest in using POMs as nano catalysts in peptide hydrolysis also the potential Keggin POM transformation in phosphate buffered saline medium was investigated leading to insight that nanoparticles of zirconium phosphate (ZrP) can be actual catalysts for breakdown of the peptide bond.

Glucomannan and beta-glucan degradation by Mytilus edulis Cel45A: Crystal structure and activity comparison with GH45 subfamily A, B and C.

The enzymatic hydrolysis of barley beta-glucan, konjac glucomannan and carboxymethyl cellulose by a ß-1,4-D-endoglucanase MeCel45A from blue mussel, Mytilus edulis, which belongs to subfamily B of glycoside hydrolase family 45 (GH45), was compared with GH45 members of subfamilies A (Humicola insolens HiCel45A), B (Trichoderma reesei TrCel45A) and C (Phanerochaete chrysosporium PcCel45A). Furthermore, the crystal structure of MeCel45A is reported. Initial rates and hydrolysis yields were determined by reducing sugar assays and product formation was characterized using NMR spectroscopy. The subfamily B and C enzymes exhibited mannanase activity, whereas the subfamily A member was uniquely able to produce monomeric glucose. All enzymes were confirmed to be inverting glycoside hydrolases. MeCel45A appears to be cold adapted by evolution, as it maintained 70% activity on cellohexaose at 4 °C relative to 30 °C, compared to 35% for TrCel45A. Both enzymes produced cellobiose and cellotetraose from cellohexaose, but TrCel45A additionally produced cellotriose.

Amylose starch with no detectable branching developed through DNA-free CRISPR-Cas9 mediated mutagenesis of two starch branching enzymes in potato.

DNA-free genome editing was used to induce mutations in one or two branching enzyme genes (Sbe) in tetraploid potato to develop starch with an increased amylose ratio and elongated amylopectin chains. By using ribonucleoprotein (RNP) transfection of potato protoplasts, a mutation frequency up to 72% was achieved. The large variation of mutations was grouped as follows: Group 1 lines with all alleles of Sbe1 mutated, Group 2 lines with all alleles of Sbe1 as well as two to three alleles of Sbe2 mutated and Group 3 lines having all alleles of both genes mutated. Starch from lines in Group 3 was found to be essentially free of amylopectin with no detectable branching and a chain length (CL) distribution where not only the major amylopectin fraction but also the shortest amylose chains were lost. Surprisingly, the starch still formed granules in a low-ordered crystalline structure. Starch from lines of Group 2 had an increased CL with a higher proportion of intermediate-sized chains, an altered granule phenotype but a crystalline structure in the granules similar to wild-type starch. Minor changes in CL could also be detected for the Group 1 starches when studied at a higher resolution.

Experimental evidence of chemical exchange over the beta(1-->3) glycosidic linkage and hydrogen bonding involving hydroxy protons in hyaluronan oligosaccharides by NMR spectroscopy.

The hydroxy protons of unsaturated di-, tetra-, hexa- and octa-saccharides of hyaluronan (DeltaHA(2), DeltaHA(4), DeltaHA(6) and DeltaHA(8)) in 85% H(2)O/15% acetone-d(6) have been studied by NMR spectroscopy. The chemical shifts (delta), chemical shift differences (Deltadelta), temperature coefficients (ddelta/dT) and nuclear or rotating-frame Overhauser effects (NOEs or ROEs) of hydroxy protons were measured to gain insight into hydration, hydrogen bonds and flexibility of the HA structure. The NMR data give the first experimental evidence that weak hydrogen bonds exist between O(4)H of N-acetyl-D-glucosamine (GlcNAc) and O(5) of D-glucuronic acid (GlcA) across the beta(1-->3) glycosidic linkage and between O(3)H of GlcA and O(5) of GlcNAc across the beta(1-->4)-linkage. A chemical exchange was observed between O(4)H of GlcNAc and O(2)H of GlcA over the beta(1-->3)-linkage. The interaction could be mediated through water bridge(s) and thus contribute to the water-retaining ability of hyaluronan. In this study it was also demonstrated how the chemical shifts of exchangeable hydroxy or amide proton signals can be used to describe small structural and conformational perturbations within large oligosaccharides.

Band-selective NMR experiments for suppression of unwanted signals in complex mixtures.

Band-selective NMR experiments are presented that allow selective suppression of unwanted signals (SUN) from the spectra of complex metabolite mixtures. As a result, spectral overlap and dynamic range problems are substantially reduced and low-intensity signals normally covered by dominant signals can be observed. The usefulness of the experiments is exemplified with selective suppression of sugar signals from the NMR spectra of fruit juice and a plant sample. Other possible applications include blood, milk, and wine samples.

Relaxation and diffusion of water protons in BDDE cross-linked hyaluronic acid hydrogels investigated by NMR spectroscopy-Comparison with physicochemical properties.

Cross-linked hyaluronic acid (HA) hydrogels are used in many biomedical applications but their characterization in order to distinguish between physicochemical properties is challenging. Longitudinal (T1) and transverse (T2) relaxation times and diffusion coefficient (D) of water protons in diepoxide 1,4-butanediol diglycidyl ether (BDDE)-cross-linked HA hydrogels were analyzed by high-field NMR spectroscopy to distinguish between different physicochemical properties. Hydrogels of different degrees of modification and cross-linking, representing a range of gel content, swelling ability, elastic and viscous behavior were studied, as well as solutions of native HA of different molecular weights. T1, T2 and D were measured for several concentrations of HA and as a function of temperature. D and T1 showed a weak concentration dependence, but did not differ between the hydrogels. T2, dominated by chemical exchange between water protons and exchangeable protons of HA, varied significantly between the different hydrogels and the temperature profiles changed dramatically between different concentrations.

NMR study of hydroxy and amide protons in hyaluronan polymers.

Hyaluronan (HA) is an important and well characterized glycosaminoglycan with high viscosity and water-retaining capacity. Nonetheless, it is not fully understood whether conformational properties of the easily characterized HA oligomers can be transferred to HA polymers. To investigate possible differences in hydration, hydrogen bonding and flexibility between HA polymers and oligomers, hydroxy and amide protons of HA polymers were studied by solution-state and high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Measurements of chemical shifts, temperature coefficients and NOEs in HA polymers revealed that the NMR data are very similar compared to the interior of a HA octasaccharide, supporting transient hydrogen bond interactions across the ß(1→3) and ß(1→4) glycosidic linkages. However, differences in NOEs suggested a cis-like orientation between NH and H2 in the HA polymer. The lack of concentration dependence of the hydroxy proton chemical shifts suggests that there are no direct inter-chain interactions involving hydroxy protons at the concentrations investigated.

Synthesis and tentative identification of novel polybrominated diphenyl ether metabolites in human blood.

Hydroxylated polybrominated diphenyl ethers (OH-PDBEs) are exogenous, bioactive compounds that originate, to a large extent, from anthropogenic activities, although they are also naturally produced in the environment. In the present study nine new authentic OH-PBDE reference standards and their corresponding methyl ether derivatives (MeO-PBDEs) were synthesised and characterised by NMR spectroscopy and mass spectrometry. Seven of the authentic reference standards prepared were thereafter tentatively identified in a pooled human blood sample. The tentatively identified OH-PBDEs were 3-hydroxy-2,2',4,4',6-pentabromodiphenyl ether, 3'-hydroxy-2,2',4,4',6-pentabromodiphenyl ether, 3-hydroxy-2,2',4,4',5-pentabromodiphenyl ether, 3-hydroxy-2,2',4,4',5,6'-hexabromodiphenyl ether, 3'-hydroxy-2,2',4,4',5,6'-hexabromodiphenyl ether, 3-hydroxy-2,2',4,4',5,5'-hexabromodiphenyl ether and 4-hydroxy-2,2',3,4',5,5',6-heptabromodiphenyl ether. An additional seven OH-PBDEs were tentatively identified in the pooled human blood sample, of which one OH-PBDE, 4'-hydroxy-2,2',4,5,5'-pentabromodiphenyl ether, has not been identified in human blood before. The identification was performed using gas chromatography-mass spectrometry (GC-MS) recording the bromine ions m/z 79, 81. The tentative identification was supported by the peaks relative retention times (RRTs) compared to authentic references on two GC columns of different polarities for the hexa-, and heptabrominated OH-PBDEs, and three different GC columns for the pentabrominated OH-PBDEs. The OH-PBDE congeners most likely originate from human metabolism of a flame retardant, i.e. polybrominated diphenyl ethers (PBDEs), due to the relatively high concentrations of PBDEs in the same human blood sample and the fact that these PBDEs could form the tentatively identified OH-PBDEs via metabolic direct hydroxylation or via 1,2-shift.

High-resolution ¹H magic angle spinning NMR spectroscopy of intact Arctic char (Salvelinus Alpinus) muscle. Quantitative analysis of n-3 fatty acids, EPA and DHA.

The lipid and small metabolite profiles from intact muscles of Arctic char were investigated using (1)H high-resolution magic angle spinning ((1)H HR-MAS) NMR spectroscopy. Not only the total n-3 fatty acid content but also the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) contents of the muscle were obtained from the (1)H HR-MAS NMR spectra without pretreatment of the tissue or lipophilic extraction. A number of small metabolites could also be observed, where creatine/phosphocreatine, anserine and taurine were the most abundant. Thus, the use of (1)H HR-MAS NMR led to simplified analysis techniques that can give direct information on the nutritional value of the fish.