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.     

Mass spectrometry‐based metabolomics towards understanding of gene functions with a diversity of biological contexts

Currently, mass spectrometry‐based metabolomics studies extend beyond conventional chemical categorization and metabolic phenotype analysis to understanding gene function in various biological contexts (e.g., mammalian, plant, and microbial). These novel utilities have led to many innovative discoveries in the following areas: disease pathogenesis, therapeutic pathway or target identification, the biochemistry of animal and plant physiological and pathological activities in response to diverse stimuli, and molecular signatures of host–pathogen interactions during microbial infection. In this review, we critically evaluate the representative applications of mass spectrometry‐based metabolomics to better understand gene function in diverse biological contexts, with special emphasis on working principles, study protocols, and possible future development of this technique. Collectively, this review raises awareness within the biomedical community of the scientific value and applicability of mass spectrometry‐based metabolomics strategies to better understand gene function, thus advancing this application's utility in a broad range of biological fields. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:118–128, 2013     

Bacterial endotoxins in periodontal health and diseases

Bacterial endotoxins are a major concern in periodontal health and diseases owing to their structure and biological activity. With up-to-date knowledge of endotoxins and the recent findings about the influence of endotoxins in dental health, their probable mode of pathogenesis, and standard detection methods, this review analyzes the potential efficacy and benefits of probiotics in combination with conventional and contemporary treatment measures. In the oral cavity, Gram-negative bacteria are documented to predominate in the pulpal lesions with radiolucent areas and in the root canal with pulp necrosis, where they pose an absolute threat by promoting a series of inflammatory reactions. Endotoxin, a constituent of Gram-negative bacteria establishes a nexus between cytokine stimulation and proinflammatory reactions, therefore plays a critical role in decaying dental pulp and modulating periodontal diseases. Currently, the treatment regimen involves several biochemical preparations. In addition, probiotics have been reported to control endotoxin in gingivitis and contribute to the overall improvement of dental health. A potential benefit of a combination of probiotics as a complementary treatment along with the conventional treatment warrant more empirical evidence to elucidate its role and mechanism in resolving the clinical manifestations associated with endotoxins in the periodontal region.     

Bacteria-hemocyte interactions and phagocytosis in marine bivalves

Marine bivalves (such as mussels, oysters, and clams) are widespread mollusks in coastal waters at different latitudes; due to their filter-feeding habits, they accumulate large numbers of bacteria from the harvesting waters and may act as passive carriers of human pathogens. To cope with this challenge, bivalves possess both humoral and cellular defense mechanisms with remarkably effective capabilities. The circulating cells, or hemocytes, are primarily responsible for defense against parasites and pathogens; microbial killing results from the combined action of the phagocytic process with humoral defense factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), toxic oxygen intermediates, and various antimicrobial peptides. In this work, current knowledge of the mechanisms underlying the interactions between bacteria and the hemolymph components of marine bivalves is summarized. Bacterial susceptibility to hemolymph killing in different bivalve species may be a consequence of the different ability of bacterial products to attract phagocytes, the presence or absence of specific opsonizing molecules, the hemocyte capability to bind and engulf different bacteria, and the different bacterial sensitivity to intracellular killing. The role of soluble (e.g., agglutinins and opsonins) and surface-bound factors in bacterial phagocytosis by hemocytes of the most common marine bivalve species is described and the possibility that environmental temperatures and other seasonal factors may influence this process is considered. Moreover, the potential strategies used by bacteria to evade phagocytic killing by hemocytes are discussed. From the available data it is clear that several questions need further investigation; the elucidation of the factors influencing phagocytosis in bivalves and the fundamental strategies used by bacteria to escape hemolymph killing are important not only to understand bivalve immune defenses but also to explain the persistence of pathogenic bacteria in bivalve tissues and to predict the consequent impact on human health.     

Mass spectrometry-based metabolomics

This review presents an overview of the dynamically developing field of mass spectrometry-based metabolomics. Metabolomics aims at the comprehensive and quantitative analysis of wide arrays of metabolites in biological samples. These numerous analytes have very diverse physico-chemical properties and occur at different abundance levels. Consequently, comprehensive metabolomics investigations are primarily a challenge for analytical chemistry and specifically mass spectrometry has vast potential as a tool for this type of investigation. Metabolomics require special approaches for sample preparation, separation, and mass spectrometric analysis. Current examples of those approaches are described in this review. It primarily focuses on metabolic fingerprinting, a technique that analyzes all detectable analytes in a given sample with subsequent classification of samples and identification of differentially expressed metabolites, which define the sample classes. To perform this complex task, data analysis tools, metabolite libraries, and databases are required. Therefore, recent advances in metabolomics bioinformatics are also discussed.     

UPLC/Q-TOFMSE法快速鉴定奋乃静在人胆汁中的代谢物

The metabolites of perphenazine in human bile were rapid investigated employing UPLC/Q-TOF MS^E (where E represents energy) technique. The bile samples were collected after dosing to human and purified through acetonitrile protein precipitation. Data collection was done by UPLC/Q-TOF MS^E. According to the molecule formular educed from the measured accurate mass, mass defect filter(MDF) technique and generic dealkylation tool were used to screen the metabolites. Their structures were elicited by comparing the fragmentation patterns between parent drug and metabolites. All the metabolites has chlorine atom isotope cluster. After oral administration of perphenazine to the patient, 29 metabolites including 16 phase I and 13 phase II metabolites are detected in human bile. The major metabolic pathways of perphenazine in human are hydroxylation, N-dealkylation, desaturation, sulfation and glucuronate conjugation.     

奋乃静在人胆汁中的代谢物分析

建立了超高效液相色谱 四极杆串联飞行时间质谱（UPLC/Q-TOF MS）检测并表征奋乃静在人胆汁中代谢物的方法。T管收集一名精神病患者服用奋乃静后的胆汁样品,经乙腈沉淀蛋白预处理后,采用UPLC/Q-TOF MS进行分析。根据高分辨质谱给出的准确分子质量信息推测可能的分子式,结合MS E功能采集的前体离子和产物离子信息,利用质量缺损过滤（MDF）和generic dealkylation等代谢物鉴定软件筛选代谢物。通过对比原形药物和代谢物的质谱裂解途径,推测可能的代谢物结构。在服药（4 mg,b.i.d）后的人胆汁中,共检测到29种奋乃静代谢物,包括I相代谢物16种,II相代谢物13种,其中16种为首次报道的新颖代谢物。奋乃静在人体内的主要代谢途径包括羟基化、脱氢、N-去烷基化、甲基化、硫酸及葡萄糖醛酸结合等,该结果进一步完善了奋乃静在人体内的代谢途径。     

Advances in mass spectrometry for the identification of pathogens

Mass spectrometry (MS) has become an important technique to identify microbial biomarkers. The rapid and accurate MS identification of microorganisms without any extensive pretreatment of samples is now possible. This review summarizes MS methods that are currently utilized in microbial analyses. Affinity methods are effective to clean, enrich, and investigate microorganisms from complex matrices. Functionalized magnetic nanoparticles might concentrate traces of target microorganisms from sample solutions. Therefore, nanoparticle-based techniques have a favorable detection limit. MS coupled with various chromatographic techniques, such as liquid chromatography and capillary electrophoresis, reduces the complexity of microbial biomarkers and yields reliable results. The direct analysis of whole pathogenic microbial cells with matrix-assisted laser desorption/ionization MS without sample separation reveals specific biomarkers for taxonomy, and has the advantages of simplicity, rapidity, and high-throughput measurements. The MS detection of polymerase chain reaction (PCR)-amplified microbial nucleic acids provides an alternative to biomarker analysis. This review will conclude with some current applications of MS in the identification of pathogens.     

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.     

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。     

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法的最新进展,包括微纳液相色谱、多维色谱与高分辨质谱联用、直接进样高分辨质谱、高分辨质谱成像技术及代谢物定性策略等,并对其发展前景进行展望。     

光谱技术在食源性致病菌检测中的应用

如何快速准确地对食源性致病菌进行检测,是控制食品安全问题的关键。传统的生化培养方法操作繁琐、费时费力,无法满足食源性致病菌快速检测的需求。随着光谱技术的发展,已经建立了不少快速、简便、特异、敏感、低耗的食源性致病菌检测新技术。阐述了食源性致病菌光谱分析的最新研究和发展趋势,展望了弹性散射光谱技术在单细胞水平实时、快速、无标记自动识别单个菌体细胞的应用,此平台的搭建将推动食源性致病菌无损检测技术的发展。     

基于离子淌度-质谱技术分析小分子代谢物的研究进展

近年来,不同原理的离子淌度技术相继出现,与质谱技术相结合已广泛应用于许多领域。在小分子代谢物分析中,氨基酸的手性识别、聚糖的结构解析、脂质的结构表征和类固醇的分析十分重要,但由于小分子代谢物化学性质迥异,且普遍存在同分异构现象,增加了分析难度。离子淌度-质谱(IM-MS)技术为复杂基质中小分子代谢物的快速分离和分析提供了新思路,根据离子的质荷比、碰撞截面积(CCS)和结构信息,可快速区分、表征小分子代谢物及其同分异构体。本文介绍了离子淌度-质谱(IM-MS)的主要类型及其在小分子代谢物分析中的应用情况和存在的不足,并展望其发展前景。     

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.     

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.     

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。     

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。     

Environmental scanning electron microscopy investigations of biodeterioration

Case studies will be presented in which environmental scanning electron microscopy (ESEM) has been used to provide unique insight into the role of microorganisms in deterioration processes. ESEM is an excellent tool for demonstrating spatial relationships between microorganisms and substrata because hydrated, nonconducting samples can be viewed with a minimum of manipulation. Copper and iron-rich deposits associated with bacteria were detected within corrosion layers on copper and steel surfaces, respectively. Fungal mycelia growing on wooden storage spools were shown to penetrate protective grease on carbon steel wire rope in contact with the spool and to cause localized corrosion. Large numbers of marine bacteria were documented within paint blisters and disbonded regions of fiber-reinforced polymeric composites. In both cases, it appears that microbial gas production resulted in mechanical damage to the substrata.     

生物流体驱动系统及其仿生

大自然一直启发着人类在工业上的成就,仿生学越来越多地应用于流体驱动技术。对拥有流体驱动系统的生物相关研究进行了介绍,并总结了利用流体实现优美敏捷动作的机理。总结得出生物流体驱动系统的存在需要3个特征:动力源、腔体和工作介质。其中动力源类似于液压泵,腔体类似于液压缸,工作介质类似于液压油。对生物流体驱动系统的深入研究和探索,可以为解决流体驱动系统使用面临的挑战提供参考;并探讨了流体驱动系统仿生应用与未来发展的新思路,促进使用生物启发的方法,改善工程流体驱动系统。     

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.