Application of Two‐Dimensional NMR Spectroscopy to Metabotyping Laboratory Escherichia coli Strains

NMR Spectroscopy has been established as a major tool for identification and quantification of metabolites in a living system. Since the metabolomics era began, one‐dimensional NMR spectroscopy has been intensively employed due to its simplicity and quickness. However, it has suffered from an inevitable overlap of signals, thus leading to inaccuracy in identification and quantification of metabolites. Two‐dimensional (2D) NMR has emerged as a viable alternative because it can offer higher accuracy in a reasonable amount of time. We employed ¹H,¹³C‐HSQC to profile metabolites of six different laboratory E. coli strains. We identified 18 metabolites and observed clustering of six strains according to their metabolites. We compared the metabolites among the strains, and found that a) the strains specialized for protein production were segregated; b) XL1‐Blue separated itself from others by accumulating amino acids such as alanine, aspartate, glutamate, methionine, proline, and lysine; c) the strains specialized for cloning purpose were spread out from one another; and d) the strains originating from B strain were characterized by succinate accumulation. This work shows that 2D‐NMR can be applied to identify a strain from metabolite analysis, offering a possible alternative to genetic analysis to identify E. coli strains.     

An introduction to biological nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful analytical techniques available to biology. This review is an introduction to the potential of this method and is aimed at readers who have little or no experience in acquiring or analyzing NMR spectra. We focus on spectroscopic applications of the magnetic resonance effect, rather than imaging ones, and explain how various aspects of the NMR phenomenon make it a versatile tool with which to address a number of biological problems. Using detailed examples, we discuss the use of ¹H NMR spectroscopy in mixture analysis and metabolomics, the use of ¹³C NMR spectroscopy in tracking isotopomers and determining the flux through metabolic pathways (‘fluxomics’) and the use of ³¹P NMR spectroscopy in monitoring ATP generation and intracellular pH homeotasis in vivo. Further examples demonstrate how NMR spectroscopy can be used to probe the physical environment of a cell by measuring diffusion and the tumbling rates of individual metabolites and how it can determine macromolecular structures by measuring the bonds and distances which separate individual atoms. We finish by outlining some of the key challenges which remain in NMR spectroscopy and we highlight how recent advances—such as increased magnet field strengths, cryogenic cooling, microprobes and hyperpolarisation—are opening new avenues for today's biological NMR spectroscopists.     

NMR metabolomic of frontal cortex extracts: First study comparing two neurodegenerative diseases,Alzheimer disease and amyotrophic lateral sclerosis

ObjectiveThis study was designed to assess the brain metabolites’ variability between two neurodegenerative diseases in frontal cortex samples obtained post-mortem. NMR metabolomics was used for the first time in this context.Materials and methods¹H NMR metabolomic was applied to tissue extracts from patients with Alzheimer disease (ALZ) and patients with amyotrophic lateral sclerosis (ALS) to investigate qualitative and quantitative variations of brain metabolites.ResultsThe Alzheimer disease metabolic signature was characterized by a high concentration of alanine, acetate, glutamate and glutamine, and low concentrations of lactate and creatine, while the ALS metabolic signature appears to be marked by high concentrations of lactate, N-acetyl aspartate, creatine, choline and myo-inositol. Moreover, in vitro ¹H NMR could detect metabolites such as 3-hydroxybutyrate, alanine, succinate and aspartate that cannot be detected with in vivo NMR.DiscussionThe neurodegenerative diseases exhibit diverging metabolic pathways. Some of the metabolites responsible for the discrimination between the two diseases were detected before in vivo. However, this in vitro metabolomic investigation demonstrates the involvement of metabolites not detected with in vivo studies.ConclusionUpon these findings, in vitro metabolomics appears to be an efficient tool to investigate the fundamentals of the metabolic pathway modulations in these neurodegenerative diseases to help the interpretation of clinical data obtained with in vivo NMR spectroscopy.     

Investigation of species and environmental effects on rhubarb roots metabolome using <Superscript>1</Superscript>H NMR combined with high performance thin layer chromatography

Introduction

The pharmacological activities of medicinal plants are reported to be due to a wide range of metabolites, therein, the concentrations of which are greatly affected by many genetic and/or environmental factors. In this context, a metabolomics approach has been applied to reveal these relationships. The investigation of such complex networks that involve the correlation between multiple biotic and abiotic factors and the metabolome, requires the input of information acquired by more than one analytical platform. Thus, development of new metabolomics techniques or hyphenations is continuously needed.

Objectives

Feasibility of high performance thin-layer chromatography (HPTLC) were investigated as a supplementary tool for medicinal plants metabolomics supporting ¹H nuclear magnetic resonance (¹H NMR) spectroscopy.

Method

The overall metabolic difference of plant material collected from two species (Rheum palmatum and Rheum tanguticum) in different geographical locations and altitudes were analyzed by ¹H NMR- and HPTLC-based metabolic profiling. Both NMR and HPTLC data were submitted to multivariate data analysis including principal component analysis and orthogonal partial least square analysis.

Results

The NMR and HPTLC profiles showed that while chemical variations of rhubarb are in some degree affected by all the factors tested in this study, the most influential factor was altitude of growth. The metabolites responsible for altitude differentiation were chrysophanol, emodin and sennoside A, whereas aloe emodin, catechin, and rhein were the key species-specific markers.

Conclusion

These results demonstrated the potential of HTPLC as a supporting tool for metabolomics due to its high profiling capacity of targeted metabolic groups and preparative capability.

     

Influence of ethanol concentration of extraction solvent on metabolite profiling for Salviae Miltiorrhizae Radix et Rhizoma extract by 1H NMR spectroscopy and multivariate data analysis

Salviae Miltiorrhizae Radix et Rhizoma (Danshen in China) and its related preparations are widely used in clinical practice due to its high medicinal value. In recent years, ¹H NMR technology has made great progress and demonstrated its unique advantages in the field of botanical metabolomics. In this study, ¹H NMR-based metabolomics was used to investigate the dissolution of various metabolites in Danshen as a function of ethanol concentration. ¹H NMR spectroscopy of Danshen extract identified 28 metabolites including 6 sugars, 11 amino acids, 3 organic acids, 4 salvianolic acids, and 4 tanshinones. Multivariate statistical analysis was used to classify and compare various Danshen extracts. PCA and HCA were used to obtain a global overview of the similarity in the samples and two-class OPLS-DA models were established for identifying characteristic metabolites. Then, ¹H-qNMR method was used to estimate the concentration of 22 metabolites, which is helpful to further describe the changes in metabolite ratios of various Danshen extracts. The result of this study laid the foundation for further biological activity research, and also provided an important reference for subsequent process research and quality control of Danshen related preparations.     

NMR analysis of plant nitrogen metabolism

The analysis of primary and secondary nitrogen metabolism in plants by nuclear magnetic resonance (NMR) spectroscopy is comprehensively reviewed. NMR is a versatile analytical tool, and the combined use of ¹H, ²H, ¹³C, ¹⁴N and ¹⁵N NMR allows detailed investigation of the acquisition, assimilation and metabolism of nitrogen. The analysis of tissue extracts can be complemented by the in vivo NMR analysis of functioning tissues and cell suspensions, and by the application of solid state NMR techniques. Moreover stable isotope labelling with ²H-, ¹³C- and ¹⁵N-labelled precursors provides direct insight into specific pathways, with the option of both time-course and steady state analysis increasing the potential value of the approach. The scope of the NMR method, and its contribution to studies of plant nitrogen metabolism, are illustrated with a wide range of examples. These include studies of the GS/GOGAT pathway of ammonium assimilation, investigations of the metabolism of glutamate, glycine and other amino acids, and applications to tropane alkaloid metabolism. The continuing development of the NMR technique, together with potential applications in the emerging fields of metabolomics and metabolic flux analysis, leads to the conclusion that NMR will play an increasingly valuable role in the analysis of plant nitrogen metabolism.     

Identification of metabolites from 2D 1H-13C HSQC NMR using peak correlation plots

Background

Identification of individual components in complex mixtures is an important and sometimes daunting task in several research areas like metabolomics and natural product studies. NMR spectroscopy is an excellent technique for analysis of mixtures of organic compounds and gives a detailed chemical fingerprint of most individual components above the detection limit. For the identification of individual metabolites in metabolomics, correlation or covariance between peaks in ¹H NMR spectra has previously been successfully employed. Similar correlation of 2D ¹H-¹³C Heteronuclear Single Quantum Correlation spectra was recently applied to investigate the structure of heparine. In this paper, we demonstrate how a similar approach can be used to identify metabolites in human biofluids (post-prostatic palpation urine).

Results

From 50 ¹H-¹³C Heteronuclear Single Quantum Correlation spectra, 23 correlation plots resembling pure metabolites were constructed. The identities of these metabolites were confirmed by comparing the correlation plots with reported NMR data, mostly from the Human Metabolome Database.

Conclusions

Correlation plots prepared by statistically correlating ¹H-¹³C Heteronuclear Single Quantum Correlation spectra from human biofluids provide unambiguous identification of metabolites. The correlation plots highlight cross-peaks belonging to each individual compound, not limited by long-range magnetization transfer as conventional NMR experiments.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0413-z) contains supplementary material, which is available to authorized users.     

Suitability of dried herbarium specimens for NMR metabolomics of mushrooms. A comparison of four species of the genera Kuehneromyces and Hypholoma (Strophariaceae)

Herbarium specimens are a treasure trove for biochemical studies. However, this implies understanding of the chemical changes during the drying and storage of the specimen. We compared herbarium specimens at different ages and fresh samples of four mushroom species (Kuehneromyces mutabilis, Hypholoma capnoides, Kuehneromyces lignicola, Hypholoma fasciculare) of two genera in the family Strophariaceae by using proton nuclear magnetic resonance (¹H NMR) spectroscopy combined with principal component analysis (PCA). 25 metabolites were identified. No significant alterations were found between herbarium samples at different ages, suggesting that they are stable enough for comparative studies. The most dominant differences between fresh and herbarium samples was that sugars such as α-α-trehalose, and fumaric and malic acids were more abundant in fresh fungi. Total contents of fatty and amino acids, uracil and γ-aminobutyric acid (GABA) were higher in herbarium specimens. In addition, pyroglutamic acid was observed only in Kuehneromyces mutabilis and fasciculic acid E in Hypholomafasciculare. Hence, based on results of the studied taxa, we conclude that NMR metabolomics can be used for both fresh and dried mushrooms when such alterations are properly addressed.     

Can Urine Metabolomics Be Helpful in Differentiating Neuropathic and Nociceptive Pain? A Proof-of-Concept Study

The diagnosis of pain nature is a troublesome task and a wrong attribution often leads to an increase of costs and to avoidable pharmaceutical adverse reactions. An objective and specific approach to achieve this diagnosis is highly desirable. The aim of this work was to investigate urine samples collected from patients suffering from pain of different nature by a metabolomics approach based on ¹H NMR spectroscopy and multivariate statistical analysis. We performed a prospective study on 74 subjects: 37 suffering from pain (12 with nociceptive and 25 with neuropathic pain), and 37 controls not suffering from any kind of chronic pain. The application of discriminant analysis on the urine spectral profiles allowed us to classify these two types of pain with high sensibility and specificity. Although the classification relies on the global urine metabolic profile, the individual contribution in discriminating neuropathic pain patients of metabolites such as choline and phosphocholine, taurine and alanine, suggests potential lesions to the nervous system. To the best of our knowledge, this is the first time that a urine metabolomics profile is used to classify these two kinds of pain. This methodology, although based on a limited sample, may constitute the basis for a new helpful tool in the clinical diagnosis.     

NMR-based metabolomics at work in phytochemistry

Metabolomics is defined as both the qualitative and quantitative analysis of all metabolites in an organism unraveling correlation with other OMICs data. Many of the technologies used in metabolomics have method-specific advantages and drawbacks in terms of diversity of metabolites detected, sensitivity, or resolution. In this paper, the potential of NMR spectrometry applied to metabolomics is reviewed using examples of Nicotiana tabacum and Catharanthus roseus.     

Microbial metabolomics: past,present and future methodologies

Microbial metabolomics has received much attention in recent years mainly because it supports and complements a wide range of microbial research areas from new drug discovery efforts to metabolic engineering. Broadly, the term metabolomics refers to the comprehensive (qualitative and quantitative) analysis of the complete set of all low molecular weight metabolites present in and around growing cells at a given time during their growth or production cycle. This review focuses on the past, current and future development of various experimental protocols in the rapid developing area of metabolomics in the ongoing quest to reliably quantify microbial metabolites formed under defined physiological conditions. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites as well as quantification of these metabolites using enzyme based and or modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (LC-MSⁿ, GC-MSⁿ, CE-MSⁿ), where n indicates the number of tandem mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR).     

生态代谢组学研究进展

代谢组学指某一生物系统中产生的或已存在的代谢物组的研究,以质谱和核磁共振技术为分析平台,以信息建模与系统整合为目标。随着代谢组学中的研究方法与技术成为生态学研究的有力工具,生态代谢组学概念应运而生,即研究某一个生物体对环境变化的代谢物组水平的响应。理清代谢组学与生态代谢组学学科发展的脉络,综述代谢组学研究中的常用技术及其优势与局限性,论述代谢组学技术在生态学研究中的应用现状,展望代谢组学技术与其他系统生物学组学技术的结合在生态学中的应用前景,提出生态代谢组学研究者未来要完成的任务和面对的挑战。     

NMR-derived developmental metabolic trajectories: an approach for visualizing the toxic actions of trichloroethylene during embryogenesis

Fish embryo toxicity tests for chemical risk assessment have traditionally been based upon non-specific endpoints including morphological abnormalities, hatching success, and mortality. Here we extend the application of ¹H NMR-based metabolomics in environmental toxicology by adding a suite of metabolic endpoints to the Japanese medaka (Oryzias latipes) embryo assay, with the goal to provide more sensitive, specific and unbiased biomarkers of toxicity. Medaka were exposed throughout embryogenesis to five concentrations of trichloroethylene (TCE; 0, 8.76, 21.9, 43.8, 87.6, 175 mg/L) and the relative sensitivities of the traditional and metabolomic endpoints compared. While the no-observable-adverse-effect-level for hatching success, the most sensitive traditional indicator, was 164 mg/L TCE, metabolic perturbations were detected at all exposure concentrations. Principal components analysis (PCA) highlighted a dose-response relationship between the NMR spectra of medaka extracts. In addition, 12 metabolites that exhibited highly significant dose-response relationships were identified, which indicated an energetic cost to TCE exposure. Next, embryos were exposed to 0, 0.88, 8.76 mg/L TCE and sampled on each of the 8 days of development. Projections of 66 two-dimensional J-resolved NMR spectra were obtained, and PCA revealed developmental metabolic trajectories that characterized the basal and TCE-perturbed changes in the entire NMR-visible metabolome throughout embryogenesis. Although no significant increases in mortality, gross deformity or developmental retardation were observed relative to the control group, TCE-induced metabolic perturbations were observed on day 8. In conclusion, these results support the continued development of NMR-based metabolomics as a rapid and reproducible tool for biomarker discovery and environmental risk assessment.     

Discrimination and Characterization of Two Mediterranean Species from the Laurencia Complex (Rhodomelacea) Using an NMR‐Based Metabolomic Approach

Generic and specific determination among the Laurencia complex is a challenging task. DNA barcoding combined with phenotypic investigations are mandatory for species differentiation. In this study, two morphologically different members of the Laurencia complex were investigated using untargeted ¹H‐NMR‐based metabolomics. Twenty‐one population samples were collected in order to evaluate both temporal and geographical homogeneity. Data obtained from ¹H‐NMR analysis followed by statistical analysis allowed a clear separation of all the samples into two groups. DNA mitochondrial tests confirmed this pattern and identified the two species as Laurenciella sp. and Laurencia obtusa. In addition, metabolites responsible of this discrimination were investigated directly in crude extracts by ¹³C‐NMR using an in‐house computer‐assisted method. The combination of both untargeted (¹H) and targeted (¹³C) NMR‐based metabolomic approaches proves to be a powerful and complementary approach to discriminate species from the Laurencia complex.     

A fast NMR method for resonance assignments: application to metabolomics

We present a new method for rapid NMR data acquisition and assignments applicable to unlabeled (¹²C) or ¹³C-labeled biomolecules/organic molecules in general and metabolomics in particular. The method involves the acquisition of three two dimensional (2D) NMR spectra simultaneously using a dual receiver system. The three spectra, namely: (1) G-matrix Fourier transform (GFT) (3,2)D [¹³C, ¹H] HSQC–TOCSY, (2) 2D ¹H–¹H TOCSY and (3) 2D ¹³C–¹H HETCOR are acquired in a single experiment and provide mutually complementary information to completely assign individual metabolites in a mixture. The GFT (3,2)D [¹³C, ¹H] HSQC–TOCSY provides 3D correlations in a reduced dimensionality manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete ¹H and ¹³C assignments of a mixture of 21 unlabeled metabolites corresponding to a medium used in assisted reproductive technology. Taken together, the experiments provide time gain of order of magnitudes compared to the conventional data acquisition methods and can be combined with other fast NMR techniques such as non-uniform sampling and covariance spectroscopy. This provides new avenues for using multiple receivers and projection NMR techniques for high-throughput approaches in metabolomics.     

Metabolic profiling of gender: Headspace-SPME/GC–MS and <Superscript>1</Superscript>H NMR analysis of urine

This study aims to investigate the metabolic difference between male and female healthy adults using a combination of GC–MS and NMR metabolomics techniques. While metabolomics has shown wide applications in characterizing the status and progression of many diseases, physiological factors such as gender often contribute high levels of variability that can hinder the detection of biomarkers of interest, such as in disease detection. We carried out a detailed exploration of gender related metabolic profiling of human urine using a Headspace-SPME/GC–MS approach and detected over two hundred peaks. Fifty-nine metabolites were identified using the NIST library. ¹H NMR spectroscopy was also utilized, and resulted in the identification of eighteen metabolites. We find that both GC–MS and NMR are able to capture human gender metabolic differences, and their combination allows a significantly better understanding of this difference. Subtle differences between genders are found to be related to the metabolism of fats, amino acids, and TCA cycle intermediates.     

高分辨魔角旋转核磁共振和主成分分析研究人类低级星形细胞瘤和脑膜瘤的代谢组特征

采用高分辨魔角旋转核磁共振（HRMAS ^1H NMR）技术结合主成分分析（PCA）方法研究了39例人体脑肿瘤组织的代谢组特征．39例肿瘤样本分别来自39个脑肿瘤患者,包括15例低级星形细胞瘤,13例纤维型脑膜瘤和11例过渡型脑膜瘤．核磁共振波谱分析结果表明,脑肿瘤组织的代谢组中丰要含有脂肪酸、乳酸、胆碱代谢物（如胆碱、磷酸胆碱和甘油磷酸胆碱）、氯基酸（如丙氨酸、谷氨酸、谷氮酰胺、牛磺酸）、N-乙酰天门冬氨酸（NAA）和谷胱甘肽等代谢物．通过对核磁共振谱进行主成分分析（PCA）,发现低级星形细胞瘤和脑膜瘤的代谢组之间具有明显的差异,而在过渡型和纤维型两个亚类脑膜瘤之间该差别相对较小．与脑膜瘤相比,低级星形细胞瘤中甘油磷酸胆碱、磷酸胆碱、肌醇与肌酸的含量较高,而丙氨酸、谷氨酸、谷氨酰胺、谷胱甘肽和牛磺酸的含量较低．NAA的含量在低级星形细胞瘤中尽管较低但能观察到,而脑膜瘤中却未发现NAA的信号．结果衷明,HRMAS ^1H NMR和多变量统计分析相结合的组织代谢组学方法,不仅能有效区分不同类型的脑肿瘤,而且还可以为脑肿瘤提供丰富的代谢组信息,这些信息对研究肿瘤发生发展的机制具有潜在的意义．     

1H-NMR-based metabolomics for cancer targeting and metabolic engineering –A review

Nuclear magnetic resonance (NMR) spectroscopy acts as the best tool that can be used in tissue engineering scaffolds to investigate unknown metabolites. Moreover, metabolomics is a systems approach for examining in vivo and in vitro metabolic profiles, which promises to provide data on cancer metabolic alterations. However, metabolomic profiling allows for the activity of small molecules and metabolic alterations to be measured. Furthermore, metabolic profiling also provides high-spectral resolution, which can then be linked to potential metabolic relationships. An altered metabolism is a hallmark of cancer that can control many malignant properties to drive tumorigenesis. Metabolite targeting and metabolic engineering contribute to carcinogenesis by proliferation, and metabolic differentiation. The resulting the metabolic differences are examined with traditional chemometric methods such as principal component analysis (PCA), and partial least squares-discriminate analysis (PLS-DA). In this review, we examine NMR-based activity metabolomic platforms that can be used to analyze various fluxomics and for multivariant statistical analysis in cancer. We also aim to provide the reader with a basic understanding of NMR spectroscopy, cancer metabolomics, target profiling, chemometrics, and multifunctional tools for metabolomics discrimination, with a focus on metabolic phenotypic diversity for cancer therapeutics.     

Qualitative high field <Superscript>1</Superscript>H-NMR spectroscopy for the characterization of endogenous metabolites in earthworms with biochemical biomarker potential

This study was designed to provide a database of the endogenous metabolites in earthworm extracts of the species, Eisenia veneta and Lumbricus terrestris by high resolution ¹H-NMR spectroscopy in view of identifying biomarkers of toxicity or stress in environmental metabolomics studies. 1D and 2D NMR spectroscopic techniques enabled the identification and confirmation of the organic components in the tissue extracts of whole and segmented earthworms, dissected organs, and coelomic fluid. The extracts gave rise to characteristic ¹H-NMR spectral fingerprints of the low MW metabolites contained, specific to the species of earthworm, and to the specific regions or dissected organs of the earthworms under investigation. Distinct changes in the normal biochemistry were observed upon starvation and cooling, such as markedly decreased glucose and maltose, but increased lactate, acetate, succinate, formate and acetone. Additionally, slightly decreased threonine, arginine, lysine, leucine, citrate, asparagine and glycine were observed. Furthermore, lactate could be identified as a biomarker of acute toxic stress in expressed coelomic fluid following exposure to a model ecotoxin (3-trifluoromethylaniline). This work supports the application of ¹H-NMR spectroscopy for the study of changes in the normal invertebrate biochemistry in order to allow for the reliable assessment of biomarker responses following toxicity testing.