款冬花不同发育阶段的代谢组学和比较转录组学分析

以不同发育阶段款冬花（发育初期、中期、中后期、解封期及花朵期）为对象,基于核磁共振的代谢组学技术,分析其次生代谢物种类及含量的合成累积规律,并进行高通量转录组学测序,从差异表达的基因中寻找次生代谢物生物合成的关联酶基因。代谢组学分析发现,款冬花不同发育阶段的次生代谢物代谢组成明显不同,苯丙素类成分在发育初期至中后期含量较高,之后逐渐降低;黄酮类成分（芦丁、山奈酚）在发育的各个阶段含量均有波动,但总体变化不大。转录组学分析结果显示,与苯丙素类成分生物合成相关的酶基因（COMT、HCT）,随着花蕾的发育表达量逐渐降低;与黄酮类成分合成相关的酶基因（FLS、F3H、DFR）,其表达量在不同阶段变化不大。转录组测序中的相关酶基因表达量同次生代谢物成分的变化趋势基本一致。本文采用的代谢组学和转录组学结合的方式,对款冬花的次生代谢物累积规律进行分析,为今后款冬花次生代谢物的生物合成调控研究奠定了基础。     

基于质谱技术的细胞代谢组学研究进展

细胞代谢组学作为一个新兴领域,能解决基本的生物学问题,还能观察细胞内的代谢情况。细胞代谢物浓度可以近似地反映一个组织、器官或细胞的表型。随着代谢组学的发展,以质谱分析为基础的代谢组学技术研究细胞的代谢物,其灵敏度高、分辨率好,能进行多组分的检测,并能获取分子的结构信息,这有利于细胞生物学的研究。该文结合目前代谢组学的技术,对细胞代谢物研究的意义及基于质谱技术的细胞代谢组学的应用进行了综述。     

植物应答非生物胁迫的代谢组学研究进展

代谢组学技术是研究植物代谢的理想平台, 通过现代检测分析技术对胁迫环境下植物中代谢产物进行定性和定量分析, 可以监测其随时间变化的规律。而各种组学平台包括基因组学、转录组学及代谢组学的整合, 更是一个强有力的工具箱, 将所获得的不同组学的信息联系起来, 有利于从整体研究生物系统对基因或环境变化的响应, 如可判断代谢物的变化是从哪一个层面开始发生的, 帮助人们揭开复杂的植物胁迫应答机制。该文对近期代谢组学技术及其与蛋白质组学、基因组学技术相结合探索植物应答非生物胁迫的研究进行了综述。代谢组学的应用, 拓展了对植物耐受非生物胁迫分子机制的认识, 开展更多这方面的研究, 再通过植物代谢组学、转录组学、蛋白质组学和基因组学整合, 有助于从整体水平上把握植物胁迫应答机制。     

基于代谢组学技术的植物抗病相关代谢物研究进展

植物受到病原真菌侵染时往往通过调节体内代谢物的产生来增强自身抗性,代谢组学技术是研究植物抗病相关代谢物的重要工具。指认植物抗病相关代谢物不仅利于深入探讨其抗病机制,还可与其他组学技术结合,辅助抗性品种鉴定和抗病品种培育。该研究对近年来国内外有关基于代谢组学技术指认植物抗病相关代谢物的流程、已发现的抗病相关代谢物及其作用机制的研究进展进行综述,并探讨了目前应用代谢组学技术研究植物抗病相关代谢物过程中面临的挑战。     

柞蚕蛹培养蛹虫草不同时间后的代谢组分析

为了解柞蚕蛹培养蛹虫草（简称柞蚕蛹虫草）不同时间后的代谢物变化规律,利用广泛靶向代谢组学技术,比较不同培养时期的柞蚕蛹虫草代谢产物成分,找出差异代谢物并进行代谢通路分析。在柞蚕蛹虫草的5个生长时期共检测到10类421种化合物,主要包括：氨基酸及其衍生物、核苷酸及其衍生物、萜类、酚酸、有机酸、糖及醇类、甾体、脂类、生物碱、醌类等多种化合物。主成分分析（PCA）结果显示,不同生长时期柞蚕蛹虫草的代谢组表现出不同的代谢特征,5个生长时期样本在得分图中可分为3个不同区域,对照（S1）与子座形成初期（S3）归为一区,菌核期（S2）独自归为一区,子座形成期（S4）和子囊壳形成期（S5）归为一区。S2-S5筛选到的特有差异代谢物有36种,包括虫草素、甘露醇等主要活性物质,其中香豆酰腐胺、五羟色胺、γ-生育三烯酚等物质首次被检测到。对不同生长阶段变化排在前20的差异显著代谢成分进行分析,结果表明上下调幅度较大的物质多为有机酸、酚酸、生物碱、核苷酸及其衍生物等次生代谢产物。5个生长时期筛选出的所有差异代谢物共富集在154条代谢通路上,差异代谢物数量富集最多的前5条通路,分别为代谢途径、次生代谢物的生物合成、ABC转运蛋白、嘌呤代谢和氨酰生物合成。整个生长周期中,具有显著影响的通路是嘌呤代谢与丙氨酸、天门冬氨酸和谷氨酸代谢途径。本研究结果可为柞蚕蛹虫草的深入研究与应用提供参考。     

代谢组学技术及其在临床研究中的应用

在后基因组时代, 系统生物学研究成为人们关注的焦点。转录组学、蛋白质组学等功能基因组学研究方法可同时检测药物或其他因素影响下大量基因或蛋白质的表达变化情况, 但这些变化不能与生物学功能的变化建立直接联系。代谢组学方法则可为代谢物含量变化与生物表型变化建立直接相关性。代谢组学研究的目的是定量分析一个生物系统内所有代谢物的含量, 进行全面代谢物分析需要分析化学技术的支撑, 核磁共振和基于质谱的分析技术是代谢组学研究的两种主要技术手段。代谢组学研究可产生大量数据信息, 对这些数据进行分析离不开化学统计学的应用, 比如主成分分析、多维缩放、各种聚类分析技术以及功能差异分析等。文章综述了近年来代谢组学分析技术及数据分析技术的研究进展, 在此基础上, 对代谢组学在临床研究及临床前研究中的应用研究进展进行了综述。对疾病代谢表型图谱的研究有助于人们了解疾病发生、发展以及致死的机制; 在临床条件下, 这些代谢图谱可以作为疾病诊断、预后以及治疗的评判标准。代谢物组成的变化是毒物胁迫对机体造成的最终影响, 利用代谢组技术可以直接反映毒物对机体的影响。质谱技术、核磁共振技术的应用使得药物筛选过程可以快速完成, 并有助于实现个性化用药。此外, 利用代谢组学技术还可以进行已知酶的新活性研究, 也可以研究未知酶。     

细胞代谢组学样品前处理研究进展

细胞代谢组学是系统生物学的重要组成部分,是对生物系统进行整体和动态的认识的科学,主要进行小分子代谢物定性和定量分析研究,观察代谢物的浓度变化,从而在细胞水平上考察代谢机制。细胞代谢组学的工作流程包括:实验设计、样品采集、样品处理、代谢物分析和数据处理。其中,样品前处理方法不尽相同,而设计一个合理方便的样品前处理方法对后期开展代谢组学至关重要。现简要综述现阶段对细胞代谢组学样品前处理的研究成果和常用方法。     

Human Metabolome Technologies实力还是未知数的代谢组公司受到关注

Human Metabo1ome Technologics（HMT）是拥有代谢组解析技术的创业企业。 所谓代谢组是指存在于生物体内或细胞内的所有代谢物。在细胞里面除了像蛋白质那样的大分子还存在有机羧酸（Carboxylic Acid）、糖磷酸化合物等低分子的代谢物。在细胞内呼吸以及光合作用过程中产生各种各样的代谢物．这些代谢物的量随时在变动。如果能够解析清楚代谢物的话．就有可能找到细胞内的基因以及蛋白质的表达解析结果体现不出来的差异。     

代谢物组学在医药研究中的成功实践

代谢物组学作为后基因时代的一种全新的组学技术。其主要以现代系统生物学为理论基础,以生物体液为研究对象,以现代谱学分析理论和生物样品制备方法为技术支撑,集中生物体内低分子量化学组分进行全息分析和海量数据挖掘,最终明晰机体生物学变化的本质。代谢物组学在功能基因组学、病理生理学、药理毒理学等方面都有着广泛的应用前景。本文以代谢物组学概念化的提出为切入点,着眼于代谢物组学的宽口径应用领域,重点概述代谢物组学在医药领域的成功实践,并对代谢物组学的未来发展做初步构想代谢物组学在功能基因组学、病理生理学、药理毒理学等方面都有着广泛的应用前景。     

基于LC-MS代谢组学技术的丽豆-大豆差异代谢物比较研究

丽豆(Calophaca sinica)是我国华北地区特有的一种珍稀野生植物。为探明丽豆的营养价值，该文以大豆(Glycine max)为参照组，利用液相色谱-质谱联用(LC-MS)技术对其种子进行了比较代谢组学研究。结果表明：(1)丽豆和大豆中共检测到1 857种代谢产物，二者成分相同且含量相似的代谢物有1 698种(>90%),差异代谢物有159种(<10%)。(2)在差异代谢物中，成分差异的有9种，其中有5种为丽豆特有，剩余150种均为含量差异，其中48种(约30%)在丽豆中的含量高于大豆。(3) KEGG注释到8条差异代谢物显著富集(P<0.1)的通路，主要包括初生代谢物的各类氨基酸生物合成途径和次生代谢物的罗汉松脂素、花生四烯酸以及二萜类等生物合成途径。(4)丽豆比大豆含量低的化学组分主要是初生代谢产物，比大豆含量高的化学组分主要是次生代谢物，而这些次生代谢物在调节血糖、骨坏损修复、增强免疫以及消炎抗癌等生理过程中有着积极的作用。综上所述，该研究认为丽豆与大豆具有相近的营养价值，并且对改善人类亚健康状况有积极的影响；此外，该文使我们对丽豆的营养价值和代谢组成...     

A multimodal metabolomics approach using imaging mass spectrometry and liquid chromatography-tandem mass spectrometry for spatially characterizing monoterpene indole alkaloids secreted from roots

Plants release specialized (secondary) metabolites from their roots to communicate with other organisms, including soil microorganisms. The spatial behavior of such metabolites around these roots can help us understand roles for the communication; however, currently, they are unclear because soil-based studies are complex. Here, we established a multimodal metabolomics approach using imaging mass spectrometry (IMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially assign metabolites under laboratory conditions using agar. In a case study using Catharanthus roseus, we showed that 58 nitrogen (N)-containing metabolites are released from the roots into the agar. For the metabolite assignment, we used ¹⁵N-labeled and non-labeled LC-MS/MS data, previously reported. Four metabolite ions were identified using authentic standard compounds as derived from monoterpene indole alkaloids (MIAs) such as ajmalicine, catharanthine, serpentine, and yohimbine. An alkaloid network analysis using dot products and spinglass methods characterized five clusters to which the 58 ions belong. The analysis clustered ions from the indolic skeleton-type MIAs to a cluster, suggesting that other communities may represent distinct metabolite groups. For future chemical assignments of the serpentine community, key fragmentation patterns were characterized using the ¹⁵N-labeled and non-labeled MS/MS spectra.     

Tandem mass spectrometry for the detection of plant pathogenic fungi and the effects of database composition on protein inferences

LC-MS/MS has demonstrated potential for detecting plant pathogens. Unlike PCR or ELISA, LC-MS/MS does not require pathogen-specific reagents for the detection of pathogen-specific proteins and peptides. However, the MS/MS approach we and others have explored does require a protein sequence reference database and database-search software to interpret tandem mass spectra. To evaluate the limitations of database composition on pathogen identification, we analyzed proteins from cultured Ustilago maydis, Phytophthora sojae, Fusarium graminearum, and Rhizoctonia solani by LC-MS/MS. When the search database did not contain sequences for a target pathogen, or contained sequences to related pathogens, target pathogen spectra were reliably matched to protein sequences from nontarget organisms, giving an illusion that proteins from nontarget organisms were identified. Our analysis demonstrates that when database-search software is used as part of the identification process, a paradox exists whereby additional sequences needed to detect a wide variety of possible organisms may lead to more cross-species protein matches and misidentification of pathogens.     

Measurement of 25-hydroxyvitamin D in the clinical laboratory: Current procedures, performance characteristics and limitations

In this review we describe procedures, performance characteristics and limitations of methods available for the measurement of 25-hydroxyvitamin (25OHD) since the year 2000. The two main types of methods are competitive immunoassay and those based on chromatographic separation followed by non-immunological direct detection (HPLC, LC-MS/MS). Lack of a reference standard for 25OHD has, until recently, been a major issue resulting in poor between-method comparability. Fortunately this should soon improve due to the recent introduction of a standard reference material in human serum (SRM 972) from the National Institute of Standards and Technology (NIST). For immunoassay, specificity can be an issue especially in relation to the proportion of 25OHD2 that is quantified whereas HPLC and LC-MS/MS methods are able to measure the two major vitamin D metabolites 25OHD2 and 25OHD3 independently. HPLC and LC-MS/MS require more expensive equipment and expert staff but this can be offset against lower reagent costs. Increasingly procedures are being developed to semi-automate or automate HPLC and LC-MS/MS but run times remain considerably longer than for immunoassays especially if performed on automated platforms. For most HPLC and LC-MS/MS methods extraction and procedural losses are corrected for by the inclusion of an internal standard which, in part, may account for higher results compared to immunoassay. In general precision of immunoassay, HPLC and LC-MS/MS are comparable and all have the required sensitivity to identify severe vitamin D deficiency. Looking to the future it is hoped that the imminent introduction of a standard reference method (or methods) for 25OHD will further accelerate improvements in between method comparability.     

Metabolomic analysis reveals reliance on secondary plant metabolites to facilitate carnivory in the Cape sundew,Drosera capensis

Background and AimsSecondary metabolites are integral to multiple key plant processes (growth regulation, pollinator attraction and interactions with conspecifics, competitors and symbionts) yet their role in plant adaptation remains an underexplored area of research. Carnivorous plants use secondary metabolites to acquire nutrients from prey, but the extent of the role of secondary metabolites in plant carnivory is not known. We aimed to determine the extent of the role of secondary metabolites in facilitating carnivory of the Cape sundew, Drosera capensis.MethodsWe conducted metabolomic analysis of 72 plants in a time-series experiment before and after simulated prey capture. We used ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) and the retention time index to identify compounds in the leaf trap tissue that changed up to 72 h following simulated prey capture. We identified associated metabolic pathways, and cross-compared these compounds with metabolites previously known to be involved in carnivorous plants across taxa.Key ResultsFor the first time in a carnivorous plant, we have profiled the whole-leaf metabolome response to prey capture. Reliance on secondary plant metabolites was higher than previously thought – 2383 out of 3257 compounds in fed leaves had statistically significant concentration changes in comparison with unfed controls. Of these, ~34 compounds are also associated with carnivory in other species; 11 are unique to Nepenthales. At least 20 compounds had 10-fold changes in concentration, 12 of which had 30-fold changes and are typically associated with defence or attraction in non-carnivorous plants.ConclusionsSecondary plant metabolites are utilized in plant carnivory to an extent greater than previously thought – we found a whole-metabolome response to prey capture. Plant carnivory, at the metabolic level, likely evolved from at least two distinct functions: attraction and defence. Findings of this study support the hypothesis that secondary metabolites play an important role in plant diversification and adaptation to new environments.     

Liquid chromatography-tandem mass spectrometric determination of ceramides and related lipid species in cellular extracts

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous qualitative and quantitative determination of sphingolipid metabolites such as ceramides, sphingisine, sphinganine, sphingomyelins, and ceramide 1-phosphates in the extracts of human promyelocytic leukemia cells (HL-60). The assay uses C(4) ceramide as an internal standard; simple liquid extraction; a short XTerra MS C(18) (3 microm, 50 mm x2.0 mm) column; a gradient mobile phase of 5mM ammonium formate (pH 4.0)/methanol/tetrahydrofuran (5/2/3-->1/2/7); mass spectrometric detection using electrospray ionization. This LC-MS/MS method allowed the identification of 22 sphingolipid derivatives at pmol levels. In addition, this technique was successfully applied to analyze the changes of the sphingolipids profiles in cancer cells treated with apoptosis inducing agents, C(2) ceramide and H(2)O(2).     

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.     

Analysis of glucosinolates, isothiocyanates, and amine degradation products in vegetable extracts and blood plasma by LC-MS/MS

Dietary glucosinolates are under intensive investigation as precursors of cancer-preventive isothiocyanates. Quantitation of the dose and bioavailability of glucosinolates and isothiocyanates requires a comprehensive analysis of the major dietary glucosinolates, isothiocyanates, and related metabolites. We report a liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) analytical method for the comprehensive analysis of the seven major dietary glucosinolates, related isothiocyanates, and putative amine degradation products. The parent glucosinolates were sinigrin, gluconapin, progoitrin, glucoiberin, glucoraphanin, glucoalyssin, and gluconasturtiin. The LC-MS/MS analysis method for these compounds was developed and validated; a standard addition analysis protocol was used generally to avoid the requirement for stable isotopic standards. Where stable isotopic standards were available, internal standardization with these gave estimates in agreement with those obtained by the standard addition analysis protocol. For glucosinolates, negative ion electrospray LC-MS/MS analysis was performed. Isothiocyanates and amines were prederivatized to the corresponding thiourea and N-acetamides, respectively, and were quantified by positive ion electrospray LC-MS/MS. The limits of detection were 0.5-2 pmol; the recoveries for glucosinolates, isothiocyanates, and amines were 85-90%, 50-85%, and 60-70%, respectively; and the intra- and interbatch coefficients of variation were 1-4% and 3-10%, respectively. These methods provide facile access to comprehensive analytical data on the major dietary glucosinolates and related metabolites to quantify inputs and metabolic formation of these compounds in cancer prevention and related studies.     

Identification of potassium dehydroandrographolidi succinas and its major metabolites in rat urine by liquid chromatography-tandem mass spectrometry

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for identification of potassium dehydroandrographolidi succinas and its metabolites in rat urine. Five male rats were administrated a single dose (100 mg/kg) of potassium dehydroandrographolidi succinas by i.v. injection. The urine were sampled from 0 to 24 h and purified by using Oasis? HLB extraction cartridge, then the purified urine samples were separated on a reversed-phase C18 column with a linear gradient and detected by an on-line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular mass (Δm) and MS/MS spectra with those of the parent drug. Seven metabolites and the parent drug were found in rat urine. All these metabolites were reported for the first time.     

Granger causality in integrated GC–MS and LC–MS metabolomics data reveals the interface of primary and secondary metabolism

Metabolomics has emerged as a key technique of modern life sciences in recent years. Two major techniques for metabolomics in the last 10 years are gas chromatography coupled to mass spectrometry (GC–MS) and liquid chromatography coupled to mass spectrometry (LC–MS). Each platform has a specific performance detecting subsets of metabolites. GC–MS in combination with derivatisation has a preference for small polar metabolites covering primary metabolism. In contrast, reversed phase LC–MS covers large hydrophobic metabolites predominant in secondary metabolism. Here, we present an integrative metabolomics platform providing a mean to reveal the interaction of primary and secondary metabolism in plants and other organisms. The strategy combines GC–MS and LC–MS analysis of the same sample, a novel alignment tool MetMAX and a statistical toolbox COVAIN for data integration and linkage of Granger Causality with metabolic modelling. For metabolic modelling we have implemented the combined GC–LC–MS metabolomics data covariance matrix and a stoichiometric matrix of the underlying biochemical reaction network. The changes in biochemical regulation are expressed as differential Jacobian matrices. Applying the Granger causality, a subset of secondary metabolites was detected with significant correlations to primary metabolites such as sugars and amino acids. These metabolic subsets were compiled into a stoichiometric matrix N. Using N the inverse calculation of a differential Jacobian J from metabolomics data was possible. Key points of regulation at the interface of primary and secondary metabolism were identified.