NMR and pattern recognition methods in metabolomics: From data acquisition to biomarker discovery: A review

Metabolomics is the discipline where endogenous and exogenous metabolites are assessed, identified and quantified in different biological samples. Metabolites are crucial components of biological system and highly informative about its functional state, due to their closeness to functional endpoints and to the organism's phenotypes. Nuclear Magnetic Resonance (NMR) spectroscopy, next to Mass Spectrometry (MS), is one of the main metabolomics analytical platforms. The technological developments in the field of NMR spectroscopy have enabled the identification and quantitative measurement of the many metabolites in a single sample of biofluids in a non-targeted and non-destructive manner. Combination of NMR spectra of biofluids and pattern recognition methods has driven forward the application of metabolomics in the field of biomarker discovery. The importance of metabolomics in diagnostics, e.g. in identifying biomarkers or defining pathological status, has been growing exponentially as evidenced by the number of published papers. In this review, we describe the developments in data acquisition and multivariate analysis of NMR-based metabolomics data, with particular emphasis on the metabolomics of Cerebrospinal Fluid (CSF) and biomarker discovery in Multiple Sclerosis (MScl).     

Metabolomic applications of electrochemistry/Mass spectrometry

Analytical techniques used for multivariate analysis of endogenous metabolites in biological systems (e.g., metabolomics, metabonomics) must be capable of accurately and selectively monitoring many known and unknown molecules that span a diverse chemical spectrum and over extremely large dynamic concentration ranges. Mass spectrometric (MS) and electrochemical array (EC-Array) detection have been widely used for multi-component analysis with applicability to low-level (fmol) metabolites. Described here are practical considerations and results obtained with the combined use of EC-Array and MS for HPLC-based multivariate metabolomic analysis. Data presented include the study of changes in rat urinary metabolite profiles associated with xenobiotic toxin exposure analyzed by HPLC using water:acetonitrile binary gradient conditions and post-column flow splitting between EC-Array and MS detectors. Results show complementary quantitative and qualitative analysis and the ability to differentiate sample groups consistent with xenobiotic-induced histopathological changes. The potential applicability of this hyphenated technique for biomarker elucidation through measurement of redox active compounds that are commonly associated with disease pathology and xenobiotic toxicity is discussed. The use of EC reactor cells in series with MS is also presented as a means of producing likely metabolites to facilitate structural elucidation and confirmation.     

CE–MS for anionic metabolic profiling: An overview of methodological developments

The efficient profiling of highly polar and charged metabolites in biological samples remains a huge analytical challenge in metabolomics. Over the last decade, new analytical techniques have been developed for the selective and sensitive analysis of polar ionogenic compounds in various matrices. Still, the analysis of such compounds, notably for acidic ionogenic metabolites, remains a challenging endeavor, even more when the available sample size becomes an issue for the total analytical workflow. In this paper, we give an overview of the possibilities of capillary electrophoresis‐mass spectrometry (CE–MS) for anionic metabolic profiling by focusing on main methodological developments. Attention is paid to the development of improved separation conditions and new interfacing designs in CE–MS for anionic metabolic profiling. A complete overview of all CE–MS‐based methods developed for this purpose is provided in table format (Table 1) which includes information on sample type, separation conditions, mass analyzer and limits of detection (LODs). Selected applications are discussed to show the utility of CE–MS for anionic metabolic profiling, especially for small‐volume biological samples. On the basis of the examination of the reported literature in this specific field, we conclude that there is still room for the design of a highly sensitive and reliable CE–MS method for anionic metabolic profiling. A rigorous validation and the availability of standard operating procedures would be highly favorable in order to make CE–MS an alternative, viable analytical technique for metabolomics.     

基于液相色谱-质谱联用技术的代谢组学分析方法研究进展

代谢组学是研究小分子代谢物的有用工具,能够直接反映生命体终端和表型信息,在精准医学和转化医学中发挥着重要作用。色谱-质谱联用技术具有灵敏度高、选择性好、动态范围宽、信息丰富等优点,已成为代谢组学研究的主要技术平台。代谢组学分析方法的创新与进展是代谢组学在各领域广泛应用的重要前提。该文综述了近5年来基于液相色谱-质谱联用技术的代谢组学分析方法取得的成果,并对目前存在的问题及发展前景给予展望。综述引用文献81篇。     

Optimization of Sample Preparation for Metabolomics Exploration of Urine,Feces, Blood and Saliva in Humans Using Combined NMR and UHPLC-HRMS Platforms

Currently, most clinical studies in metabolomics only consider a single type of sample such as urine, plasma, or feces and use a single analytical platform, either NMR or MS. Although some studies have already investigated metabolomics data from multiple fluids, the information is limited to a unique analytical platform. On the other hand, clinical studies investigating the human metabolome that combine multi-analytical platforms have focused on a single biofluid. Combining data from multiple sample types for one patient using a multimodal analytical approach (NMR and MS) should extend the metabolome coverage. Pre-analytical and analytical phases are time consuming. These steps need to be improved in order to move into clinical studies that deal with a large number of patient samples. Our study describes a standard operating procedure for biological specimens (urine, blood, saliva, and feces) using multiple platforms (¹H-NMR, RP-UHPLC-MS, and HILIC-UHPLC-MS). Each sample type follows a unique sample preparation procedure for analysis on a multi-platform basis. Our method was evaluated for its robustness and was able to generate a representative metabolic map.     

(Xeno)metabolomics for the evaluation of aquatic organism’s exposure to field contaminated water

Environmental (xeno)metabolomics offers a major advantage compared to other approaches for the evaluation of aquatic organism’s exposure to contaminated water because its allows the simultaneous profiling of the xenometabolome (chemical xenobiotics and their metabolites accumulated in an organism exposed to environmental contaminants) and the metabolome (endogenous metabolites whose levels are altered due to an external stressor). This approach has been widely explored in lab exposure experiments, however in field studies environmental (xeno)metabolomics has only started in the last years. In this review, the papers published so far that have performed different (xeno)metabolomics approaches for the evaluation of aquatic organisms exposed to contaminated water are presented, together with their main achievements, current limitations, and future perspectives. The different analytical methods applied including sample pre-treatment (considering matrix type), platforms used (Nuclear Magnetic Resonance (NMR) and low- or high-resolution Mass Spectrometry (MS or HRMS)), and the analytical strategy (target vs non-target analysis) are discussed. The application of (xeno)metabolomics to provide information of xenobiotics mixtures accumulated in exposed organisms, either in lab or field studies, as well as biomarkers of exposure and biomarkers of effect are debated, and finally, the most commonly metabolic pathways disrupted by chemical contamination are highlighted.     

High-sensitivity analyses of metabolites in biological samples by capillary electrophoresis using dynamic pH junction-sweeping

Emerging fields of biochemical research, such as metabolomics, present challenges to current separation technologies because of the large number of metabolites present in a cell and their often low (submicromolar) concentration. Although capillary electrophoresis (CE) holds great promise as the method of choice for high-resolution separations of biological samples, it suffers from poor concentration sensitivity, especially with the use of UV detection. In CE, sweeping and dynamic pH junction represent two complementary on-line focusing techniques that have been used for sensitivity enhancement of hydrophobic and weakly acidic analytes, respectively. However, the application of either the sweeping or dynamic pH junction technique alone might, in some cases, be less effective for the analysis of certain sample mixtures. Recent work in the development of a hyphenated dynamic pH junction-sweeping technique is presented as an effective on-line method of preconcentration suitable for both hydrophilic (anionic) and hydrophobic (neutral) analytes. Sensitive analyses of flavin metabolites by CE with laser-induced fluorescence (LIF) detection is demonstrated in various biological matrixes, including cell extracts of Bacillus subtilis, pooled human plasma, as well as heat-deproteinized flavoenzymes. Enhanced analyte band narrowing and improved sensitivity is achieved for flavins using dynamic pH junction-sweeping compared to either sweeping or dynamic pH junction alone. This results in over a 1200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (LOD, defined as S/N = 3) of about 4.0 x 10(-12) M. Strategies for sensitive and more comprehensive analyses of other cell metabolites, including nucleotides, coenzymes, and steroids, are also discussed when using on-line focusing techniques in conjunction with multiplexed CE and UV detection.     

CE‐MS for metabolomics: Developments and applications in the period 2016–2018

In the field of metabolomics, CE‐MS is now recognized as a strong analytical technique for the analysis of (highly) polar and charged metabolites in a wide range of biological samples. Over the past few years, significant attention has been paid to the design and improvement of CE‐MS approaches for (large‐scale) metabolic profiling studies and for establishing protocols in order to further expand the role of CE‐MS in metabolomics. In this paper, which is a follow‐up of a previous review paper covering the years 2014–2016 (Electrophoresis 2017, 38, 190–202), main advances in CE‐MS approaches for metabolomics studies are outlined covering the literature from July 2016 to June 2018. Aspects like developments in interfacing designs and data analysis tools for improving the performance of CE‐MS for metabolomics are discussed. Representative examples highlight the utility of CE‐MS in the fields of biomedical, clinical, microbial, and plant metabolomics. A complete overview of recent CE‐MS‐based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings and MS detection mode. Finally, some general conclusions and perspectives are given.     

On-line preconcentration strategies for trace analysis of metabolites by capillary electrophoresis

Analysis of low concentrations of metabolites is required for new fields of biological research, such as metabolomics. In this review, recent work in our laboratory aimed at developing improved strategies for on-line sample preconcentration of metabolites by capillary electrophoresis (CE) is presented. Dynamic pH junction, sweeping and dynamic pH junction-sweeping represent three complementary methods for electrokinetic focusing of large volumes of sample directly on-capillary. Focusing selectivity and focusing efficiency are two factors that can be used to assess the suitability of each method for different classes of metabolites. Buffer properties can be selected to enhance the focusing of specific types of metabolites based on knowledge of the analyte physicochemical properties. The application of on-line preconcentration CE for trace analysis of metabolites in real samples of interest, such as biological fluids and cellular extracts, is also demonstrated. Under optimum conditions, up to three orders of magnitude increase in concentration sensitivity can be realized for several classes of metabolites, including catecholamines, purines, nucleosides, nucleotides, amino acids, steroids and coenzymes. Recent work on hyphenating on-line preconcentration with multiplexed CE is highlighted as a promising platform for sensitive and high-throughput analyses of metabolites.     

Analytical method development and validation of anticancer agent, 5-fluorouracil,and its metabolites in biological matrices: An updated review

Abstract

5-fluorouracil (5-FU) refers to a fluorinated pyrimidine analogue that has been widely used as an anticancer agent for colon, head, and neck cancers. Detection of 5-FU and its metabolites; 5-fluorouridine and 5-fluoro-2-deoxyuridine in biological samples allows optimization of pharmacotherapy and encourages fundamental investigations of this medication. The development of accurate and reliable sample preparation, as well as analytical methods, is critical to isolate targeted analytes from complex matrices, apart from increasing detection sensitivity of analytes. With that, this paper presents a review of prior studies pertaining to chromatographic and electrophoretic methods that focused on the analysis of 5-FU and its metabolites in biological matrices such as plasma and urine. This paper concentrates on HPLC, GC and CE systems, which are the most commonly used strategies for analytical separation of 5-FU and its metabolites from samples. Detection of these antineoplastic agents at trace level demands highly sensitive and selective analytical methodologies. Application of these analytical techniques to biological matrices is reviewed with a focus on method development strategies, including types of mobile phases and background electrolytes employed in LC and CE systems.     

Training in metabolomics research. I. Designing the experiment,collecting and extracting samples and generating metabolomics data

The study of metabolism has had a long history. Metabolomics, a systems biology discipline representing analysis of known and unknown pathways of metabolism, has grown tremendously over the past 20 years. Because of its comprehensive nature, metabolomics requires careful consideration of the question(s) being asked, the scale needed to answer the question(s), collection and storage of the sample specimens, methods for extraction of the metabolites from biological matrices, the analytical method(s) to be employed and the quality control of the analyses, how collected data are correlated, the statistical methods to determine metabolites undergoing significant change, putative identification of metabolites and the use of stable isotopes to aid in verifying metabolite identity and establishing pathway connections and fluxes. The National Institutes of Health Common Fund Metabolomics Program was established in 2012 to stimulate interest in the approaches and technologies of metabolomics. To deliver one of the program's goals, the University of Alabama at Birmingham has hosted an annual 4‐day short course in metabolomics for faculty, postdoctoral fellows and graduate students from national and international institutions. This paper is the first part of a summary of the training materials presented in the course to be used as a resource for all those embarking on metabolomics research. The complete set of training materials including slide sets and videos can be viewed at http://www.uab.edu/proteomics/metabolomics/workshop/workshop_june_2015.php . Copyright © 2016 John Wiley & Sons, Ltd.     

CE-MS for metabolomics: Developments and applications in the period 2018–2020

Capillary electrophoresis-mass spectrometry (CE-MS) is now a mature analytical technique in metabolomics, notably for the efficient profiling of polar and charged metabolites. Over the past few years, (further) progress has been made in the design of improved interfacing techniques for coupling CE to MS; also, in the development of CE-MS approaches for profiling metabolites in volume-restricted samples, and in strategies that further enhance the metabolic coverage. In this article, which is a follow-up of a previous review article covering the years 2016–2018 (Electrophoresis 2019, 40, 165–179), the main (technological) developments in CE-MS methods and strategies for metabolomics are discussed covering the literature from July 2018 to June 2020. Representative examples highlight the utility of CE-MS in the fields of biomedical, clinical, microbial, plant and food metabolomics. A complete overview of recent CE-MS-based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings, and MS detection mode. Finally, some general conclusions and perspectives are given.     

CE-MS for metabolomics: Developments and applications in the period 2008-2010

This review provides an update of the state-of-the-art of CE-MS for metabolomic purposes, covering the scientific literature from July 2008 to June 2010. This review describes the different analytical aspects with respect to non-targeted and targeted metabolomics and the new technological developments used in CE-MS for metabolomics. The applicability of CE-MS in metabolomics research is illustrated by examples of the analysis of biomedical and clinical samples, and for bacterial and plant extracts. The relevant papers on CE-MS for metabolomics are comprehensively summarized in a table, including, e.g. information on sample type and pretreatment, and MS detection mode. Future considerations such as challenges for large-scale and (quantitative) clinical metabolomics studies and the use of sheathless interfacing and different ionization techniques are discussed.     

蛋白质组学和代谢组学在微生物代谢工程中的应用

构建微生物细胞工厂是化学品、生物能源以及药物分子可持续生产的可行性策略。然而，微生物的代谢复杂、调控严谨，制约着目标产物高效合成。蛋白质组学和代谢组学可以从系统生物学角度分析酶和代谢物组分，从而理解复杂的生物系统，为微生物代谢工程改造提供重要线索。该文介绍了蛋白质组学和代谢组学在微生物代谢工程中的应用，包括基因组尺度代谢模型构建、菌株生物合成优化、指导菌株耐受性改造、限速步骤预测、植物次级代谢途径挖掘，从而为微生物合成天然产物提供新的基因或途径。在此基础上，该文还展望了生物大数据未来的发展方向。     

Advances in mass spectrometry applied to pharmaceutical metabolomics

Metabolomics, also referred to in the literature as metabonomics, is a relatively new systems biology tool for drug discovery and development and is increasingly being used to obtain a detailed picture of a drug’s effect on the body. Metabolomics is the qualitative assessment and relative or absolute quantitative measurement of the endogenous metabolome, defined as the complement of all native small molecules (metabolites less than 1,500 Da). A metabolomics study frequently involves the comparative analysis of sample sets from a normal state and a perturbed state, where the perturbation can be of any nature, such as genetic knockout, administration of a drug, or change in diet or lifestyle. Advances in mass spectrometry (MS) technologies including direct introduction or in-line chromatographic separation modes, ionization techniques, mass analyzers, and detection methods have provided powerful tools to assess the molecular changes in the metabolome. This review focuses on advances in MS pertaining to the analytical data generation for the main metabolomics methods, namely, fingerprinting, nontargeted, and targeted approaches, as they are applied to pharmaceutical drug discovery and development.     

Current trends and challenges in sample preparation for global metabolomics using liquid chromatography–mass spectrometry

The choice of sample-preparation method is extremely important in metabolomic studies because it affects both the observed metabolite content and biological interpretation of the data. An ideal sample-preparation method for global metabolomics should (i) be as non-selective as possible to ensure adequate depth of metabolite coverage; (ii) be simple and fast to prevent metabolite loss and/or degradation during the preparation procedure and enable high-throughput; (iii) be reproducible; and (iv) incorporate a metabolism-quenching step to represent true metabolome composition at the time of sampling. Despite its importance, sample preparation is often an overlooked aspect of metabolomics, so the focus of this review is to explore the role, challenges, and trends in sample preparation specifically within the context of global metabolomics by liquid chromatography-mass spectrometry (LC-MS). This review will cover the most common methods including solvent precipitation and extraction, solid-phase extraction and ultrafiltration, and discuss how to improve analytical quality and metabolite coverage in metabolomic studies of biofluids, tissues, and mammalian cells. Recent developments in this field will also be critically examined, including in vivo methods, turbulent-flow chromatography, and dried blood spot sampling.     

Metabolomics applied in the study of emerging arboviruses caused by Aedes aegypti mosquitoes: A review

Arboviruses, such as chikungunya, dengue, yellow fever, and zika, caused by the bite of the Aedes aegypti mosquito, have been a frequent public health problem, with a high incidence of outbreaks in tropical and subtropical countries. These diseases are easily confused with a flu-like illness and present very similar symptoms, difficult to distinguish, and treat appropriately. The effects that these infections cause in the organism are fundamentally derived from complex metabolic processes. A prominent area of science that investigates the changes in the metabolism of complex organisms is the metabolomics. Metabolomics measures the metabolites produced or altered in biological organisms, through the use of robust analytical platforms, such as separation techniques hyphenated with mass spectrometry, combined with bioinformatics. This review article presents an overview of the basic concepts of metabolomics workflow and advances in this field, and compiles research articles that use this omic approach to study these arboviruses. In this context, the metabolomics is applied to search new therapies, understand the viral replication mechanisms, and access the host-virus interactions.     

Analytical detection techniques for droplet microfluidics—A review

In the last decade, droplet-based microfluidics has undergone rapid progress in the fields of single-cell analysis, digital PCR, protein crystallization and high throughput screening. It has been proved to be a promising platform for performing chemical and biological experiments with ultra-small volumes (picoliter to nanoliter) and ultra-high throughput. The ability to analyze the content in droplet qualitatively and quantitatively is playing an increasing role in the development and application of droplet-based microfluidic systems. In this review, we summarized the analytical detection techniques used in droplet systems and discussed the advantage and disadvantage of each technique through its application. The analytical techniques mentioned in this paper include bright-field microscopy, fluorescence microscopy, laser induced fluorescence, Raman spectroscopy, electrochemistry, capillary electrophoresis, mass spectrometry, nuclear magnetic resonance spectroscopy, absorption detection, chemiluminescence, and sample pretreatment techniques. The importance of analytical detection techniques in enabling new applications is highlighted. We also discuss the future development direction of analytical detection techniques for droplet-based microfluidic systems.