Factors affecting the robustness of metabolite fingerprinting using 1H NMR spectra

1H NMR spectroscopy is one of the techniques whose potential is currently being explored in the emerging field of metabolomics. It is a non-targeted method, producing signals for all proton-containing chemical species. For crude plant materials the spectra are always complex, with many signals overlapping. Hence a most suitable approach for analysing them is 'metabolite fingerprinting', which is aimed at highlighting compositional similarities and exploring the overall natural variability in a population of samples. The most commonly used method for this is principal component analysis (PCA), as it allows the whole spectral trace to be analysed and the vast quantity of information to be simplified. In this paper we investigate whether there are factors which may affect the NMR spectra in a way that subsequently decreases the robustness of the metabolite fingerprinting by PCA. Imperfections in the signal registration (i.e. inconsistency of the peak position) are generally detrimental to analysing whole traces by multivariate methods. The sources of such problems are illustrated through specially designed repeatability studies using potato and tomato samples, and the analysis of a tea dataset containing many samples. Careful sample preparation can help to limit peak shifts; for instance here by attempting to control the pH of the extracts. In addition, some compounds are susceptible to interactions affecting their chemical shifts and mathematical alignment of peaks may be necessary. Lastly factors such as resolution can also affect analyses and must be carefully adjusted. Our choice of examples aims to raise awareness of potential problems. We do not question the validity of the NMR approach, but point out those areas where special care may need to be taken.     

Spectroscopic characterization of extracts from humic and fulvic fractions: IR and1H NMR spectra

Summary Infrared and proton resonance spectra have been used to characterize fraction extracted sequentially from humic and fulvic acids by diethylether, acetone, dioxane, tetrahydrofuran, pyridine and dimethylformamide. The results showed that the same solvents extracted structurally similar components from both humic and fulvic acids. On the other hand, the spectra showed solvent-dependent differences, some being characteristic for a preponderance of aliphatic structures, others for aromatic structures.     

A rapid method for monitoring intracellular lipid production using1H NMR spectroscopy

Summary A method has been developed for rapid mondtoring of lipid production in the intact yeast cells ofRhodotorula gracilis using¹H NMR spectroscopy.     

1H NMR spectra of branched-chain cyclomaltohexaoses (alpha-cyclodextrins)

The 1H NMR spectra of seven branched alpha-cyclodextrins (alpha-CDs) were observed and analyzed in detail. They were compared with spectra of alpha-CD and amylose. Although these branched alpha-CDs consist only of alpha-D-glucose with the same alpha-(1-->4) O-glucosyl binding, aside from one exception, differences in chemical shifts of corresponding signals were significantly large. Especially, differences in the chemical shift in anomeric protons were considerably large. Subtle differences in glucosyl binding directly influences chemical shifts of these protons because anomeric protons are located adjacent to the glucosyl binding sites.     

Identification of glycine spin systems in 1H NMR spectra of proteins using multiple quantum coherences

Double-quantum filtered COSY and triple-quantum filtered COSY techniques have been compared for the tripeptide Gly-Tyr-Gly and for human lysozyme. The insertion of a triple-quantum filter in the COSY experiment leads to dramatic spectral simplification in the fingerprint region of the spectrum and permits the specific identification of glycine spin systems in the complex 1H NMR spectra of proteins. The assignment of these peaks to glycine H alpha can be confirmed using 2D double-quantum correlated spectroscopy.     

Sequential resonance assignments in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy

A sequential assignment procedure is outlined, based on two-dimensional NOE ( NOESY ) and two-dimensional J-correlated spectroscopy ( COSY ), for assigning the nonexchangeable proton resonances in NMR spectra of oligonucleotides. As presented here the method is generally applicable to right-handed helical oligonucleotides of intermediate size. We applied it to a lac operator DNA fragment consisting of d( TGAGCGG ) and d( CCGCTCA ) and obtained complete assignments for the adenine H8, guanine H8, cytosine H6 and H5, thymine H6 and 5-methyl, and the deoxyribose H1', H2', H2", H3', and H4' resonances, as well as some H5', H5" (pairwise) assignments. These assignments are required for the analysis of two-dimensional NOE and J-coupling data in terms of the solution structure of oligonucleotides.     

Assignment strategies in homonuclear three-dimensional 1H NMR spectra of proteins

The increase in dimensionality of three-dimensional (3D) NMR greatly enhances the spectral resolution in comparison to 2D NMR. It alleviates the problem of resonance overlap and may extend the range of molecules amenable to structure determination by high-resolution NRM spectroscopy. Here, we present strategies for the assignment of protein resonances from homonuclear nonselective 3D NOE-HOHAHA spectra. A notation for connectivities between protons, corresponding to cross peaks in 3D spectra, is introduced. We show how spin systems can be identified by tracing cross-peak patterns in cross sections perpendicular to the three frequency axes. The observable 3D sequential connectivities in proteins are tabulated, and estimates for the relative intensities of the corresponding cross peaks are given for alpha-helical and beta-sheet conformations. Intensities of the cross peaks in the 3D spectrum of pike III parvalbumin follow the predictions. The sequential-assignment procedure is illustrated for loop regions, extended and alpha-helical conformations for the residues Ala 54-Leu 63 of parvalbumin. NOEs that were not previously identified in 2D spectra of parvalbumin due to overlap are found.     

Liquid seaweed extracts identified using <Superscript>1</Superscript>H NMR profiles

Aqueous extracts of Ascophyllum nodosum and several other brown seaweeds are manufactured commercially and widely distributed for use on agricultural crops. The increasingly regulated international trade in such products requires that they be standardized and defined to a degree not previously required. We examined commercially available extracts using quantitative ¹H NMR and principal components analysis (PCA) techniques. Extracts manufactured over a 4-year period using the same process exhibited characteristic profiles that, on PCA, clustered as a discrete group distinct from the other commercial products examined. In addition to recognizing extracts made from different seaweeds, analysis of the ¹H spectra in the 0.35–4.70 ppm region allowed us to distinguish amongst extracts produced from the same algal species by different manufacturers. This result established that the process used to make an extract is an important variable in defining its composition. A comparison of the ¹H NMR integrals for the regions 1.0–3.0 ppm and 3.0–4.38 ppm revealed small but significant changes in the A. nodosum spectra that we attribute to seasonal variation in gross composition of the harvested seaweed. Such changes are reflected in the PCA scores plots and contribute to the scatter observed within the data point cluster observed for Acadian soluble extracts when all data are pooled. Quantitative analysis using ¹H NMR (qNMR) with a certified external standard (caffeine) showed a linear relationship with extract concentration over at least an order of magnitude (2.5–33 mg/mL; R ² > 0.97) for both spectral regions integrated. We conclude that qNMR can be used to profile (or “fingerprint”) commercial seaweed extracts and to quantify the amount of extract present relative to a suitably chosen standard. Issued as NRCC no. 42,652.     

Main-chain-directed strategy for the assignment of 1H NMR spectra of proteins

A strategy for assigning the resonances in two-dimensional (2D) NMR spectra of proteins is described. The method emphasizes the analysis of through-space relationships between protons by use of the two-dimensional nuclear Overhauser effect (NOE) experiment. NOE patterns used in the algorithm were derived from a statistical analysis of the combinations of short proton-proton distances observed in the high-resolution crystal structures of 21 proteins. One starts with a search for authentic main-chain NH-C alpha H-C beta H J-coupled units, which can be found with high reliability. The many main-chain units of a protein are then placed in their proper juxtaposition by recognition of predefined NOE connectivity patterns. To discover these connectivities, the 2D NOE spectrum is examined, in a prescribed order, for the distinct NOE patterns characteristic of helices, sheets, turns, and extended chain. Finally, the recognition of a few amino acid side-chain types places the discovered secondary structure elements within the polypeptide sequence. Unlike the sequential assignment approach, the main-chain-directed strategy does not rely on the difficult task of recognizing many side-chain spin systems in J-correlated spectra, the assignment process is not in general sequential with the polypeptide chain, and the prescribed connectivity patterns are cyclic rather than linear. The latter characteristic avoids ambiguous branch points in the analysis and imposes an internally confirmatory property on each forward step.     

Characterization of data analysis methods for information recovery from metabolic 1H NMR spectra using artificial complex mixtures

The assessment of data analysis methods in ¹H NMR based metabolic profiling is hampered owing to a lack of knowledge of the exact sample composition. In this study, an artificial complex mixture design comprising two artificially defined groups designated normal and disease, each containing 30 samples, was implemented using 21 metabolites at concentrations typically found in human urine and having a realistic distribution of inter-metabolite correlations. These artificial mixtures were profiled by ¹H NMR spectroscopy and used to assess data analytical methods in the task of differentiating the two conditions. When metabolites were individually quantified, volcano plots provided an excellent method to track the effect size and significance of the change between conditions. Interestingly, the Welch t test detected a similar set of metabolites changing between classes in both quantified and spectral data, suggesting that differential analysis of ¹H NMR spectra using a false discovery rate correction, taking into account fold changes, is a reliable approach to detect differential metabolites in complex mixture studies. Various multivariate regression methods based on partial least squares (PLS) were applied in discriminant analysis mode. The most reliable methods in quantified and spectral ¹H NMR data were PLS and RPLS linear and logistic regression respectively. A jackknife based strategy for variable selection was assessed on both quantified and spectral data and results indicate that it may be possible to improve on the conventional Orthogonal-PLS methodology in terms of accuracy and sensitivity. A key improvement of our approach consists of objective criteria to select significant signals associated with a condition that provides a confidence level on the discoveries made, which can be implemented in metabolic profiling studies.     

Metabotyping of long-lived mice using 1H NMR spectroscopy

Significant advances in understanding aging have been achieved through studying model organisms with extended healthy lifespans. Employing 1H NMR spectroscopy, we characterized the plasma metabolic phenotype (metabotype) of three long-lived murine models: 30% dietary restricted (DR), insulin receptor substrate 1 null (Irs1-/-), and Ames dwarf (Prop1df/df). A panel of metabolic differences were generated for each model relative to their controls, and subsequently, the three long-lived models were compared to one another. Concentrations of mobile very low density lipoproteins, trimethylamine, and choline were significantly decreased in the plasma of all three models. Metabolites including glucose, choline, glycerophosphocholine, and various lipids were significantly reduced, while acetoacetate, d-3-hydroxybutyrate and trimethylamine-N-oxide levels were increased in DR compared to ad libitum fed controls. Plasma lipids and glycerophosphocholine were also decreased in Irs1-/- mice compared to controls, as were methionine and citrate. In contrast, high density lipoproteins and glycerophosphocholine were increased in Ames dwarf mice, as were methionine and citrate. Pairwise comparisons indicated that differences existed between the metabotypes of the different long-lived mice models. Irs1-/- mice, for example, had elevated glucose, acetate, acetone, and creatine but lower methionine relative to DR mice and Ames dwarfs. Our study identified several potential candidate biomarkers directionally altered across all three models that may be predictive of longevity but also identified differences in the metabolic signatures. This comparative approach suggests that the metabolic networks underlying lifespan extension may not be exactly the same for each model of longevity and is consistent with multifactorial control of the aging process.     

Simplification of 1H NMR spectra of proteins by one-dimensional multiple quantum filtration

The use of multiple quantum filters for simplification and editing of one-dimensional ¹H NMR spectra of proteins is demonstrated. Three, four and five quantum-filtered spectra have been recorded. The technique is applicable to proteins of molecular weight up to at least 18,000. Examples obtained for a mixture of amino acids and for the proteins plastocyanin and myoglobin are reported. A remarkable degree of spectral editing can be achieved by judicious choice of experimental parameters.     

Investigation of anion binding to neutral lipid membranes using 2H NMR.

The binding of aqueous anions (ClO4-, SCN-, I-, and NO3-) to lipid bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated using deuterium (2H) and phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy. The ability of these anions to influence the 2H NMR quadrupole splittings of POPC, specifically labeled at the alpha or beta position of the choline head group, increased in the order NO3- much less than I- less than SCN- less than ClO4-. In the presence of these chaotropic anions, the quadrupole splitting increased for alpha-deuterated POPC and decreased for beta-deuterated POPC, indicating a progressive accumulation of negative charge at the membrane surface. Calibration of the 2H NMR quadrupole splittings with the amount of membrane-bound anion permitted binding isotherms to be generated for perchlorate, thiocyanate, and iodide, up to concentrations of 100 mM. The binding isotherms were analyzed by considering electrostatic contributions, according to the Gouy-Chapman theory, as well as chemical equilibrium contributions. For neutral POPC membranes, we obtained ion association constants of 32, 80, and 115 M-1 for iodide, thiocyanate, and perchlorate, respectively. These values increase in the order expected for a Hofmeister series of anions. We conclude that the factor determining whether a particular anion will bind to lipid bilayers is the ease with which that anion loses its hydration shell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of an artificial dimer of ribonuclease H using 1H NMR spectroscopy

Summary The protein fusion technique was applied in the synthesis of an artificial dimer of ribonuclease H (305 residues). ¹H NMR spectroscopy was used to analyze the structure of this dimer. Spectral profiles and pK_a values of the histidine residues obtained using ¹H NMR indicate that the dimer retains the secondary and tertiary structures of the intact monomer. Selective spin-lattice relaxation measurements suggest that the two monomeric units in the dimer are in tight contact. Furthermore, the 2D ¹H NMR and paramagnetic relaxation filter results show that the two monomers bind together through interactions between the N- and C-terminal sites of the linked regions.     

Paramagnetically shifted resonances in 1H NMR spectra of ribonucleotide reductase from Escherichia coli

The 400-MHz 1H NMR spectra of the subunit B2 of ribonucleotide reductase from Escherichia coli show paramagnetically shifted resonances at 24 ppm (exchangeable protons) and at 19 ppm (nonexchangeable protons). The protein contains an antiferromagnetically coupled dimeric iron center and a tyrosyl free radical. The paramagnetically shifted resonances must be due to the iron center, since they remain essentially unchanged in protein B2 with and without free radical. In analogy with recently published results for hemerythrin from Phascolopsis gouldii, which has a similar iron center, the 24-ppm resonance is suggested to arise from histidine ligands to the iron ions.     

d(TTTCCTCGCCGGAAA)的一维NMR氢谱分析

单链d(TTTCCTCGCCGGAAA)易溶于水,且由于其本身存在序列特异性,即可形成分子内“发夹”结构,本实验分别测得其在全重水(D2O)、92?O 8%H2O溶液中的一维1H谱,认为环出区域碱基质子的共振峰与其他同种质子的共振峰有明显的区别,主要表现在其共振峰会明显移向高场区。     

1H NMR metabolite fingerprinting and metabolomic analysis of perchloric acid extracts from plant tissues

Metabolite fingerprinting provides a powerful method for discriminating between biological samples on the basis of differences in metabolism caused by such factors as growth conditions, developmental stage or genotype. This protocol describes a technique for acquiring metabolite fingerprints from samples of plant origin. The preferred method involves freezing the tissue rapidly to stop metabolism, extracting soluble metabolites using perchloric acid (HClO4) and then obtaining a fingerprint of the metabolic composition of the sample using 1D 1H NMR spectroscopy. The spectral fingerprints of multiple samples may be analyzed using either unsupervised or supervised multivariate statistical methods, and these approaches are illustrated with data obtained from the developing seeds of two genotypes of sunflower (Helianthus annuus). Preparation of plant extracts for analysis takes 2-3 d, but multiple samples can be processed in parallel and subsequent acquisition of NMR spectra takes approximately 30 min per sample, allowing 24-48 samples to be analyzed in a week.     

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.     

Binding of antibiotic amphotericin B to lipid membranes: a 1H NMR study

The (1)H NMR technique was applied to study binding of AmB, an antifungal drug, to lipid membranes formed with egg yolk phosphatidylcholine. The analysis of (1)H NMR spectra of liposomes, containing also cholesterol and ergosterol (at 40 mol%), shows that AmB binds preferentially to the polar headgroups. Such a binding restricts molecular motion of the choline fragment in the hydrophilic region at the surface of liposomes but increases the segmental motional freedom in the hydrophobic core. The same effects are also observed in the sterol-containing membranes, except that the effect on the hydrophobic core was exclusively observed in the membranes containing ergosterol.     

Interaction of local anesthetics with lipid bilayers investigated by 1H MAS NMR spectroscopy

The membrane location of the local anesthetics (LA) lidocaine, dibucaine, tetracaine, and procaine hydrochloride as well as their influence on phospholipid bilayers were studied by ³¹P and ¹H magic-angle spinning (MAS) NMR spectroscopy. The ³¹P NMR spectra of the LA/lipid preparations confirmed that the overall bilayer structure of the membrane remained preserved. The relation between the molecular structure of the LAs and their membrane localization and orientation was investigated quantitatively using induced chemical shifts, nuclear Overhauser enhancement spectroscopy, and paramagnetic relaxation rates. All three methods revealed an average location of the aromatic rings of all LAs in the lipid-water interface of the membrane, with small differences between the individual LAs depending on their molecular properties. While lidocaine is placed in the upper chain/glycerol region of the membrane, for dibucaine and procaine the maximum of the distribution are slightly shifted into the glycerol region. Finally for tetracaine the aromatic ring is placed closest to the aqueous phase in the glycerol/headgroup region of the membrane. The hydrophobic side chains of the LA molecules dibucaine and tetracaine were located deeper in the membrane and showed an orientation towards the hydrocarbon core. In contrast the side chains of lidocaine and procaine are oriented towards the aqueous phase.