基于高分辨质谱的代谢组学分析技术研究进展

代谢组学研究的目标是对生物体系中所有内源小分子代谢物进行全面的定性和定量表征.由于代谢物组成复杂、种类繁多、理化性质各异、且浓度差异大,给分析工作带来了极大的挑战.高分辨质谱因具有高灵敏度、高质量分辨率和质量精度、宽动态范围等优势,已成为代谢组学研究的主流分析工具.本文综述了近5年来基于高分辨质谱的代谢组学分析技术和方...     

基于离子淌度质谱的代谢物碰撞截面积测量方法和数据库研究进展

代谢组学旨在全面系统地分析复杂生物样本中的代谢物。近年来,离子淌度质谱（IM-MS）技术快速发展,为代谢组学分析提供了强大的技术支撑。离子淌度质谱根据代谢物的化学结构进行气相分离,其衍生的碰撞截面积（CCS）可作为一种新的物理化学性质,辅助用于鉴定已知和未知代谢物的化学结构。碰撞截面积在代谢组学中的应用需要确保对其准确测量,同时需要构建高覆盖、高准确的碰撞截面积数据库。本文旨在介绍常见的不同类型商业化离子淌度质谱及其对小分子代谢物碰撞截面积测量和校正的原理,归纳目前可用于代谢组学应用的碰撞截面积数据库,并展望碰撞截面积在代谢组学中的应用。     

Metabolomics applications of FT-ICR mass spectrometry

Metabolomics, also known as Metabolic Profiling, is an emerging discipline under the umbrella concept of systems biology. The goal of metabolomics is to know and understand the concentrations and fluxes of endogenous metabolites within a living biological system under study. General tools are being developed for the rapid measurement of many metabolites in a single experiment, most of which are mass spectrometric methods. FT-ICR has unique advantages, as a mass spectrometric method, in this regard. Applications of FT-ICR to metabolomics analyses will be discussed and reviewed in the context of the single publication currently available.     

FUNCTIONAL METABOLOMICS DECIPHER BIOCHEMICAL FUNCTIONS AND ASSOCIATED MECHANISMS UNDERLIE SMALL-MOLECULE METABOLISM

Metabolism is the collection of biochemical reactions enabled by chemically diverse metabolites, which facilitate different physiological processes to exchange substances and synthesize energy in diverse living organisms. Metabolomics has emerged as a cutting-edge method to qualify and quantify the metabolites in different biological matrixes, and it has the extraordinary capacity to interrogate the biological significance that underlies metabolic modification and modulation. Liquid chromatography combined with mass spectrometry (LC/MS), as a robust platform for metabolomics analysis, has increased in popularity over the past 10 years due to its excellent sensitivity, throughput, and versatility. However, metabolomics investigation currently provides us with only phenotype data without revealing the biochemical functions and associated mechanisms. This limitation indeed weakens the core value of metabolomics data in a broad spectrum of the life sciences. In recent years, the scientific community has actively explored the functional features of metabolomics and translated this cutting-edge approach to be used to solve key multifaceted questions, such as disease pathogenesis, the therapeutic discovery of drugs, nutritional issues, agricultural problems, environmental toxicology, and microbial evolution. Here, we are the first to briefly review the history and applicable progression of LC/MS-based metabolomics, with an emphasis on the applications of metabolic phenotyping. Furthermore, we specifically highlight the next era of LC/MS-based metabolomics to target functional metabolomes, through which we can answer phenotype-related questions to elucidate biochemical functions and associated mechanisms implicated in dysregulated metabolism. Finally, we propose many strategies to enhance the research capacity of functional metabolomics by enabling the combination of contemporary omics technologies and cutting-edge biochemical techniques. The main purpose of this review is to improve the understanding of LC/MS-based metabolomics, extending beyond the conventional metabolic phenotype toward biochemical functions and associated mechanisms, to enhance research capability and to enlarge the applicable scope of functional metabolomics in small-molecule metabolism in different living organisms.     

Liquid chromatography combined with mass spectrometry for 13C isotopic analysis in life science research

Among the different disciplines covered by mass spectrometry, measurement of (13)C/(12)C isotopic ratio crosses a large section of disciplines from a tool revealing the origin of compounds to more recent approaches such as metabolomics and proteomics. Isotope ratio mass spectrometry (IRMS) and molecular mass spectrometry (MS) are the two most mature techniques for (13)C isotopic analysis of compounds, respectively, for high and low-isotopic precision. For the sample introduction, the coupling of gas chromatography (GC) to either IRMS or MS is state of the art technique for targeted isotopic analysis of volatile analytes. However, liquid chromatography (LC) also needs to be considered as a tool for the sample introduction into IRMS or MS for (13)C isotopic analyses of non-volatile analytes at natural abundance as well as for (13)C-labeled compounds. This review presents the past and the current processes used to perform (13)C isotopic analysis in combination with LC. It gives particular attention to the combination of LC with IRMS which started in the 1990's with the moving wire transport, then subsequently moved to the chemical reaction interface (CRI) and was made commercially available in 2004 with the wet chemical oxidation interface (LC-IRMS). The LC-IRMS method development is also discussed in this review, including the possible approaches for increasing selectivity and efficiency, for example, using a 100% aqueous mobile phase for the LC separation. In addition, applications for measuring (13)C isotopic enrichments using atmospheric pressure LC-MS instruments with a quadrupole, a time-of-flight, and an ion trap analyzer are also discussed as well as a LC-ICPMS using a prototype instrument with two quadrupoles.     

Development of LC/MS Techniques for Comprehensive Metabolome Analysis

Metabolomics is a rapidly evolving field for studying biological systems and discovering potential disease biomarkers. For any metabolomics application, metabolome analysis with adequate sensitivity and specificity is essential in defining the metabolome. Ideally, all metabolites present in a biological system are qualitatively and quantitatively profiled. Unfortunately, due to technical limitations, only a fraction of metabolites are currently analyzed by using techniques such as NMR and mass spectrometry (MS). Due to limited metabolome coverage, many important metabolome networks and some subdue changes in the metabolome may not be revealed with current techniques. In this presentation, several technical issues related to the development of LC/MS for enabling metabolome analysis will be discussed. Because of great diversity of chemical and physical properties of metabolites, we have been developing an isotope labeling LC/MS workflow with a goal of improving the metabolome coverage in analyzing biological samples such as human biofluids and tissue samples. Several labeling chemistries will be described to provide isotope tags to the metabolites for sensitive detection and accurate quantification. LC methods including multi-dimensional separation to separate the labeled metabolites with high efficiency will be discussed. New protocols for MS analysis, metabolite identification and quantitative data processing will be presented.     

Ion mobility mass spectrometry in the omics era: Challenges and opportunities for metabolomics and lipidomics

Researchers worldwide are taking advantage of novel, commercially available, technologies, such as ion mobility mass spectrometry (IM-MS), for metabolomics and lipidomics applications in a variety of fields including life, biomedical, and food sciences. IM-MS provides three main technical advantages over traditional LC-MS workflows. Firstly, in addition to mass, IM-MS allows collision cross-section values to be measured for metabolites and lipids, a physicochemical identifier related to the chemical shape of an analyte that increases the confidence of identification. Second, IM-MS increases peak capacity and the signal-to-noise, improving fingerprinting as well as quantification, and better defining the spatial localization of metabolites and lipids in biological and food samples. Third, IM-MS can be coupled with various fragmentation modes, adding new tools to improve structural characterization and molecular annotation. Here, we review the state-of-the-art in IM-MS technologies and approaches utilized to support metabolomics and lipidomics applications and we assess the challenges and opportunities in this growing field.     

Review on microbial metabolomics of probiotics and pathogens: Methodologies and applications

In recent years, microbial metabolomics, a new field that has attracted wide attention, provides a map of metabolic pathways and clarifies the interaction mechanism between microorganisms and hosts. Many microorganisms are found in the human intestine, oral cavity, vagina, etc. Probiotics could maintain the good health of the host, while pathogens and an imbalance of bacterial flora lead to a series of diseases of the body and mind. Metabolomics is a science for qualitative and quantitative analysis of all metabolites in an organism or biological system, which could provide key information to understand the related metabolic pathways and associated changes. This approach analyzes the final products of cellular regulatory processes, the level of which can be regarded as the ultimate response of the biological system to genetic or environmental changes. Microbial metabolomics has been widely used in different research fields, such as microbial phenotypic classification, mutant screening, metabolic pathways, microbial metabolic engineering, fermentation engineering monitoring and optimization, microbial environmental pollution, and so on. However, there are only a few reviews on microbial metabolomics of probiotics and pathogens. This review summarizes the main methodologies, including sample preparation, identification of metabolites, data processing, and analysis. Recent applications in microbial metabolomics of probiotics and pathogens are also described. This paper first summarized the research progress and application of microbial metabolomics from two aspects: probiotics and pathogenic bacteria. Probiotics and pathogenic bacteria do not exist independently most of the time; hence, these were reviewed in the research field of coexistence of probiotics and pathogenic bacteria, which was subdivided into important microbial research fields closely related to human health, including the human gut, oral cavity, food, and nutrition-related microorganisms. Then, the main problems and trends associated with microbial metabolomics are discussed.     

Eight key rules for successful data-dependent acquisition in mass spectrometry-based metabolomics

In recent years, metabolomics has emerged as a pivotal approach for the holistic analysis of metabolites in biological systems. The rapid progress in analytical equipment, coupled to the rise of powerful data processing tools, now provides unprecedented opportunities to deepen our understanding of the relationships between biochemical processes and physiological or phenotypic conditions in living organisms. However, to obtain unbiased data coverage of hundreds or thousands of metabolites remains a challenging task. Among the panel of available analytical methods, targeted and untargeted mass spectrometry approaches are among the most commonly used. While targeted metabolomics usually relies on multiple-reaction monitoring acquisition, untargeted metabolomics use either data-independent acquisition (DIA) or data-dependent acquisition (DDA) methods. Unlike DIA, DDA offers the possibility to get real, selective MS/MS spectra and thus to improve metabolite assignment when performing untargeted metabolomics. Yet, DDA settings are more complex to establish than DIA settings, and as a result, DDA is more prone to errors in method development and application. Here, we present a tutorial which provides guidelines on how to optimize the technical parameters essential for proper DDA experiments in metabolomics applications. This tutorial is organized as a series of rules describing the impact of the different parameters on data acquisition and data quality. It is primarily intended to metabolomics users and mass spectrometrists that wish to acquire both theoretical background and practical tips for developing effective DDA methods.     

Metabolomic applications of HILIC–LC–MS

Hydrophilic interaction liquid chromatography (HILIC), although not a new technique, has enjoyed a recent renaissance with the introduction of robust and reproducible stationary phases. It is consequently finding application in metabolomics studies, which have traditionally relied on the stability of reversed phases (RPs), since the biofluids analyzed are predominantly aqueous and thus contain many polar analytes. HILIC's retention of those polar compounds and use of solvents readily compatible with mass spectrometry have seen its increasing adoption in studies of complex aqueous metabolomes. This review describes the stationary phases and their features, surveys HILIC–LC–MS's role in metabolomics experiments, discusses approaches to data extraction and analysis including multivariate analysis, and reviews the literature on HILIC–MS applications in metabolomics. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:671–684, 2010     

生物样品中醇类代谢物的化学衍生化质谱分析方法研究进展

生物样品的代谢组学是近年来质谱领域的研究热点.醇类代谢物是生物样品非常重要的一类代谢物,主要包括脂肪醇、糖类、酚类、甘油酯类以及甾醇等.这些代谢物在体内承担着各种重要的生理学功能.然而,由于大多数醇类代谢物极性较低,缺乏易于离子化的基团,其在质谱领域的研究比胺类、酸类等代谢物少.化学衍生化技术通过设计靶向某官能团的有机...     

Mass spectrometry‐based holistic analytical approaches for metabolite profiling in systems biology studies

Metabonomics and metabolomics represent one of the three major platforms in systems biology. To perform metabolomics it is necessary to generate comprehensive “global” metabolite profiles from complex samples, for example, biological fluids or tissue extracts. Analytical technologies based on mass spectrometry (MS), and in particular on liquid chromatography–MS (LC–MS), have become a major tool providing a significant source of global metabolite profiling data. In the present review we describe and compare the utility of the different analytical strategies and technologies used for MS‐based metabolomics with a particular focus on LC–MS. Both the advantages offered by the technology and also the challenges and limitations that need to be addressed for the successful application of LC–MS in metabolite analysis are described. Data treatment and approaches resulting in the detection and identification of biomarkers are considered. Special emphasis is given to validation issues, instrument stability, and QA/quality control (QC) procedures. © 2011 Wiley Periodicals, Inc., Mass Spec Rev 30:884–906, 2011     

基于质谱技术的代谢组学在体外药物肝毒性评价中的研究进展

药物肝毒性是药物安全性评价的重要内容之一，研发早期对药物及其代谢产物潜在的肝毒性进行准确预测和评价，可以提高药物研发的成功率。将代谢组学技术与体外细胞模型相结合，以细胞代谢表型的变化为指标直接反映药物的毒性效应及毒性机制，能够改善临床前药物肝毒性的预测准确性，在药物肝毒性筛选研究中极具应用价值和发展潜力。本文综述了目前肝毒性研究中的细胞模型与培养方法，介绍了三维细胞培养模型在体外研究中的优势，并总结了基于质谱技术的代谢组学研究在体外细胞模型中的分析策略及其在药物肝毒性评价中的应用，其中基于质谱成像技术的空间分辨代谢组学方法在体外细胞模型研究中具有独特优势，有望发展成为体外肝毒性研究的有力工具。     

STRATEGIES AND CHALLENGES IN METHOD DEVELOPMENT AND VALIDATION FOR THE ABSOLUTE QUANTIFICATION OF ENDOGENOUS BIOMARKER METABOLITES USING LIQUID CHROMATOGRAPHY‐TANDEM MASS SPECTROMETRY

Metabolomics is a dynamically evolving field, with a major application in identifying biomarkers for drug development and personalized medicine. Numerous metabolomic studies have identified endogenous metabolites that, in principle, are eligible for translation to clinical practice. However, few metabolomic‐derived biomarker candidates have been qualified by regulatory bodies for clinical applications. Such interruption in the biomarker qualification process can be largely attributed to various reasons including inappropriate study design and inadequate data to support the clinical utility of the biomarkers. In addition, the lack of robust assays for the routine quantification of candidate biomarkers has been suggested as a potential bottleneck in the biomarker qualification process. In fact, the nature of the endogenous metabolites precludes the application of the current validation guidelines for bioanalytical methods. As a result, there have been individual efforts in modifying existing guidelines and/or developing alternative approaches to facilitate method validation. In this review, three main challenges for method development and validation for endogenous metabolites are discussed, namely matrix effects evaluation, alternative analyte‐free matrices, and the choice of internal standards (ISs). Some studies have modified the equations described by the European Medicines Agency for the evaluation of matrix effects. However, alternative strategies were also described; for instance, calibration curves can be generated in solvents and in biological samples and the slopes can be compared through ratios, relative standard deviation, or a modified Stufour suggested approaches while quantifying mainly endogenous metabolitesdent t‐test. ISs, on the contrary, are diverse; in which seven different possible types, used in metabolomics‐based studies, were identified in the literature. Each type has its advantages and limitations; however, isotope‐labeled ISs and ISs created through isotope derivatization show superior performance. Finally, alternative matrices have been described and tested during method development and validation for the quantification of endogenous entities. These alternatives are discussed in detail, highlighting their advantages and shortcomings. The goal of this review is to compare, apprise, and debate current knowledge and practices in order to aid researchers and clinical scientists in developing robust assays needed during the qualification process of candidate metabolite biomarkers. © 2019 John Wiley & Sons Ltd. Mass Spec Rev     

Imaging mass spectrometry

Imaging mass spectrometry combines the chemical specificity and parallel detection of mass spectrometry with microscopic imaging capabilities. The ability to simultaneously obtain images from all analytes detected, from atomic to macromolecular ions, allows the analyst to probe the chemical organization of a sample and to correlate this with physical features. The sensitivity of the ionization step, sample preparation, the spatial resolution, and the speed of the technique are all important parameters that affect the type of information obtained. Recently, significant progress has been made in each of these steps for both secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) imaging of biological samples. Examples demonstrating localization of proteins in tumors, a reduction of lamellar phospholipids in the region binding two single celled organisms, and sub-cellular distributions of several biomolecules have all contributed to an increasing upsurge in interest in imaging mass spectrometry. Here we review many of the instrumental developments and methodological approaches responsible for this increased interest, compare and contrast the information provided by SIMS and MALDI imaging, and discuss future possibilities.     

Evaluating and minimizing batch effects in metabolomics

Determining metabolomic differences among samples of different phenotypes is a critical component of metabolomics research. With the rapid advances in analytical tools such as ultrahigh-resolution chromatography and mass spectrometry, an increasing number of metabolites can now be profiled with high quantification accuracy. The increased detectability and accuracy raise the level of stringiness required to reduce or control any experimental artifacts that can interfere with the measurement of phenotype-related metabolome changes. One of the artifacts is the batch effect that can be caused by multiple sources. In this review, we discuss the origins of batch effects, approaches to detect interbatch variations, and methods to correct unwanted data variability due to batch effects. We recognize that minimizing batch effects is currently an active research area, yet a very challenging task from both experimental and data processing perspectives. Thus, we try to be critical in describing the performance of a reported method with the hope of stimulating further studies for improving existing methods or developing new methods.     

基于质谱技术的黄芩治疗2型糖尿病的粪便代谢组学研究

采用粪便代谢组学方法,运用超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF MS）技术研究了黄芩治疗2型糖尿病大鼠的作用机制。采用主成分分析（PCA）和正交偏最小二乘法判别分析方法 （OPLS-DA）对健康对照组、2型糖尿病模型组和黄芩治疗组的大鼠粪便中内源性代谢物进行分析,寻找黄芩治疗2型糖尿病大鼠的潜在生物标志物。结果表明,健康对照组、2型糖尿病模型组和黄芩治疗组的大鼠粪便代谢图谱有显著的区分;发现并鉴定了11种潜在的生物标志物。黄芩对2型糖尿病大鼠的鞘脂类代谢和脂肪酸代谢具有调节作用;对三羟基三甲基吲哚酮、白三烯E4、亮氨酰脯氨酸和雌二醇的含量具有调节作用;同时,大鼠体重和空腹血糖的变化趋势表明,黄芩具有改善糖尿病症状的作用。     

Mass spectrometric based approaches in urine metabolomics and biomarker discovery

Urine metabolomics has recently emerged as a prominent field for the discovery of non‐invasive biomarkers that can detect subtle metabolic discrepancies in response to a specific disease or therapeutic intervention. Urine, compared to other biofluids, is characterized by its ease of collection, richness in metabolites and its ability to reflect imbalances of all biochemical pathways within the body. Following urine collection for metabolomic analysis, samples must be immediately frozen to quench any biogenic and/or non‐biogenic chemical reactions. According to the aim of the experiment; sample preparation can vary from simple procedures such as filtration to more specific extraction protocols such as liquid‐liquid extraction. Due to the lack of comprehensive studies on urine metabolome stability, higher storage temperatures (i.e. 4°C) and repetitive freeze‐thaw cycles should be avoided. To date, among all analytical techniques, mass spectrometry (MS) provides the best sensitivity, selectivity and identification capabilities to analyze the majority of the metabolite composition in the urine. Combined with the qualitative and quantitative capabilities of MS, and due to the continuous improvements in its related technologies (i.e. ultra high‐performance liquid chromatography [UPLC] and hydrophilic interaction liquid chromatography [HILIC]), liquid chromatography (LC)‐MS is unequivocally the most utilized and the most informative analytical tool employed in urine metabolomics. Furthermore, differential isotope tagging techniques has provided a solution to ion suppression from urine matrix thus allowing for quantitative analysis. In addition to LC‐MS, other MS‐based technologies have been utilized in urine metabolomics. These include direct injection (infusion)‐MS, capillary electrophoresis‐MS and gas chromatography‐MS. In this article, the current progresses of different MS‐based techniques in exploring the urine metabolome as well as the recent findings in providing potentially diagnostic urinary biomarkers are discussed. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:115–134, 2017.