基于RRLC-MS/MS方法的恶性肿瘤代谢组学研究

采用基于LC-MS/MS技术的代谢组学方法,寻找恶性肿瘤患者尿液中的内源性小分子生物标志物,是具有前沿性和实用性的研究课题.LC-MS/MS技术具有高灵敏度,可以对尿液中微量代谢物进行定性与定量分析,并寻找与鉴定出新的内源性小分子生物标志物,可最终建立诊断指标.同时,将化学计量学方法用于大量、多维的谱学数据分析,充分挖掘数据中的潜在信息和内在联系,有利于发现小分子生物标志物,并将其与生物体的生物学特性和临床特征进行关联,力求阐明其生物学意义.     

汞离子对细胞代谢通路影响的代谢组学

利用超高效液相色谱-四级杆飞行时间质谱(UPLC-Q-TOF-MS)技术对汞离子作用后细胞的代谢组学进行了研究,结合化学计量学方法,对代谢组学数据进行了多维统计分析.结果表明,与非汞离子作用组相比,汞离子作用组存在能量、磷脂、脂肪酸及氨基酸代谢异常,并发现16种有显著差异的生物标记物.进一步探讨了汞中毒的细胞代谢机理及细胞的应激保护.本研究建立的基于UPLC-Q-TOF-MS技术的细胞代谢组学快速分析方法可以对细胞在重金属作用后的代谢物变化进行轮廓分析.     

化学计量学方法在脂质组学数据解析中的应用

脂质组学是依赖于分析技术而发展的一门新兴学科,用于全面表征与基因调控、蛋白表达、脂质代谢密切相关的脂质分子,揭示脂质在各种生命活动中的作用机制和代谢途径网络。随着质谱及其联用技术进一步发展和完善,脂质组学逐渐向快速、自动化和高通量的方向发展,而大规模的脂质组数据分析已成为脂质组学研究领域的一大难点。化学计量学主要应用于脂质组学中的基线校正和背景扣除、信号峰识别、同位素分布解析、统计分析等过程,因此,基于化学计量学方法的脂质组学数据自动化解析策略成为研究者关心的热点。该文对近年来化学计量学在脂质组学数据解析中的应用进行了综述,并对基于化学计量学的脂质组学数据解析的未来发展进行了展望。     

尿液代谢组学方法研究人参总皂苷治疗糖尿病心肌病大鼠作用机制

利用基于质谱的代谢组学方法考察了人参总皂苷(TG)治疗糖尿病心肌病(DCM)大鼠的效应机制;建立了糖尿病心肌病大鼠模型,并连续12周口服人参总皂苷,采用快速高分辨液相色谱/四级杆-飞行时间/质谱(RRLC/Q-TOF/MS)技术对糖尿病心肌病模型组(DCM组)和人参总皂苷治疗组(TG组)大鼠尿样的尿液代谢物进行分析,采用主成分分析(PCA)对两组代谢物进行分类,并寻找潜在生物标记物,同时检测心肌病理超微结构、血液生化指标和心肌氧化应激水平。RRLC/Q-TOF/MS检测结果表明,DCM组和TG组大鼠的尿液代谢物谱能得到很好的区分,发现并鉴定了3种生物标记物。TG降低了DCM大鼠心肌超微结构损伤并改善其血脂、血糖及心肌氧化应激水平,代谢组学研究结果表明:作用机制可能是TG对柠檬酸循环、脂肪酸代谢和氧化应激水平的调节作用。     

微生物代谢组学的样品前处理

微生物代谢组学是指全面分析(定性和定量)细胞生长或生产周期某一时刻细胞内和细胞周围的所有低分子量代谢物.微生物代谢组学研究需要可靠和重现地分析细胞内较宽动力学浓度范围(nmol～mmol)、化学功能各异的代谢产物,因此,需要对从生物量培养、灭活和代谢产物的提取到代谢物的定量分析这整个实验过程提出耐用、可重现和可靠的实验方案.本文介绍了灭活过程中细胞内代谢产物泄漏的处理方法,较为详细地论述了通过灭活草案捕获代谢反应活性方法和代谢物提取方法的优化,并对微生物代谢组学样品前处理的目前发展趋势提出初步见解.     

基于快速高分辨液相色谱串联质谱技术的代谢组学尿液分析方法的建立

采用快速高分辨液相色谱(RRLC)分离系统与QTRAP型及QTOF型MS/MS仪联用技术,通过考察尿液样本前处理方法,优化液相色谱条件和质谱检测参数,建立了用于尿液中代谢物分析的RRLC-MS方法.采用本方法对尿液浓度下的20种代表性代谢物进行了检测,考察了方法的灵敏度和精密度,证明本方法适用于尿液代谢组学的研究.对穿...     

代谢组学应用于糖肾方治疗糖尿病肾病的疗效评价

建立了基于超高效液相色谱-飞行时间质谱(UPLC/TOF MS)分析技术的血浆代谢指纹谱,应用多元统计分析方法评价糖尿病肾病患者血浆代谢物变化差异及糖肾方的干预效果。通过研究糖肾方干预糖尿病肾病血浆内源性代谢物的变化,探索与该疾病密切相关的代谢途径,评价糖肾方的治疗效果。结果发现: 经糖肾方治疗后,血浆内源性代谢物发生了明显变化,磷脂代谢、脂肪酸代谢、氨基酸代谢、嘌呤嘧啶代谢、固醇类代谢等多个代谢途径得到纠正。本研究基于UPLC/TOF MS的代谢组学方法,能够从整体水平反映疾病治疗过程中代谢网络的变化趋势,证实糖肾方具有治疗糖尿病肾病的临床疗效并有助于阐释药物作用机理。     

基于修饰组和探针分子的重要途径代谢物筛选和注释新方法北大核心CSCD

植物次生代谢物在抵御生物/非生物胁迫、生物间互作以及信息传递等方面发挥重要作用,次生代谢途径解析对植物分子育种、天然产物合成等方面具有重要意义。液相色谱-高分辨串联质谱(LC-HRMS/MS)为次生代谢物鉴定及途径表征提供了技术手段。非靶向LC-HRMS/MS方法可获得丰富的质谱信号,包括一级质谱和二级质谱(MS,MS/MS),但受质谱数据库规模以及次生代谢物复杂性的制约,次生代谢物注释十分困难。该研究以玉米叶片中苯丙烷途径代谢物为例,发展用于非靶向代谢组数据中重要途径代谢物的高效筛选和注释新方法。首先,利用公共代谢途径数据库及文献获取参与苯丙烷代谢途径的61种修饰反应类型,进而从非靶向实验数据中筛选出修饰代谢组。其次,获取开源串联质谱数据中的苯丙烷类化合物作为探针分子,构建探针分子质谱数据库。将探针分子与修饰代谢组共建分子网络,锁定目标途径代谢物并注释结构。该方法在正、负离子模式下分别筛选出玉米叶片中392个和417个苯丙烷途径候选代谢物,去冗余后共注释出129个代谢物,涉及苯丙烷代谢的主要分支途径,如黄酮途径的8个类黄酮、19个氧苷类黄酮和32个碳苷类黄酮,31个羟基肉桂酸途径代谢物以及22个木脂素途径代谢物;其中26个在PubChem和SciFinder数据库中未见收录。该研究利用探针分子结合修饰组可快速锁定途径代谢物,且有助于快速、准确的网络传播注释,可显著提高目标途径代谢物筛选与注释效率,为植物次生代谢途径的深入解析提供分析手段。     

基于液相色谱-质谱联用技术的肺癌细胞代谢组学分析

通过高效液相色谱-四极杆飞行时间质谱(HPLC-Q-TOF/MS)分别对肺癌细胞与正常细胞的极性与非极性代谢物进行指纹图谱分析,进一步应用偏最小二乘判别分析(PLS-DA)对代谢组学数据进行多维统计分析。研究结果显示,与正常细胞相比,肿瘤细胞存在异常的蛋白质、脂肪酸、磷脂代谢,并发现31种对分类有显著贡献的代谢小分子物质。通过本研究,建立了基于液相色谱-质谱联用技术的肺癌细胞代谢组学分析方法,发现了肺癌潜在疾病标记物,可为肺癌分子标记物的发现及其早期诊断提供新思路和新方法。     

基于超高效液相色谱-离子阱-飞行时间质谱联用的白血病细胞及耐药细胞非靶标代谢组学研究

建立了一种基于超高效液相色谱-离子阱-飞行时间质谱(UPLC-IT-TOF MS)联用的非靶标代谢组学方法,研究了人慢性髓系白血病细胞及其阿霉素耐药细胞(K562和K562-ADM)之间的代谢物差异。考察了细胞破碎方法、提取溶剂体系和溶剂体积对细胞代谢物提取效果的影响,实验发现采用超声破碎法,以800μL的80%甲醇提取时可得到较多的代谢特征峰,方法重复性和稳定性较高。将建立的方法应用于K562及K562-ADM细胞的代谢组差异研究,对所得数据进行主成分分析(PCA)和正交偏最小二乘法判别分析(OPLS-DA)。结果显示2种细胞的代谢特征存在显著差异,并发现40个对分类有显著贡献的代谢物,差异代谢物主要参与氨基酸代谢、鞘脂代谢、嘌呤代谢和嘧啶代谢等通路。该方法可用于K562和K562-ADM细胞的代谢组差异研究,可望为白血病细胞耐药性的代谢组学研究提供帮助。     

Automated software-guided identification of new buspirone metabolites using capillary LC coupled to ion trap and TOF mass spectrometry

The identification and structure elucidation of drug metabolites is one of the main objectives in in vitro ADME studies. Typical modern methodologies involve incubation of the drug with subcellular fractions to simulate metabolism followed by LC-MS/MS or LC-MS(n) analysis and chemometric approaches for the extraction of the metabolites. The objective of this work was the software-guided identification and structure elucidation of major and minor buspirone metabolites using capillary LC as a separation technique and ion trap MS(n) as well as electrospray ionization orthogonal acceleration time-of-flight (ESI oaTOF) mass spectrometry as detection techniques.Buspirone mainly underwent hydroxylation, dihydroxylation and N-oxidation in S9 fractions in the presence of phase I co-factors and the corresponding glucuronides were detected in the presence of phase II co-factors. The use of automated ion trap MS/MS data-dependent acquisition combined with a chemometric tool allowed the detection of five small chromatographic peaks of unexpected metabolites that co-eluted with the larger chromatographic peaks of expected metabolites. Using automatic assignment of ion trap MS/MS fragments as well as accurate mass measurements from an ESI oaTOF mass spectrometer, possible structures were postulated for these metabolites that were previously not reported in the literature.     

Metabonomics investigation of human urine after ingestion of green tea with gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry and (1)H NMR spectroscopy

A method using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and (1)H NMR with pattern recognition tools such as principle components analysis (PCA) was used to study the human urinary metabolic profiles after the intake of green tea. From the normalized peak areas obtained from GC/MS and LC/MS and peak heights from (1)H NMR, statistical analyses were used in the identification of potential biomarkers. Metabolic profiling by GC/MS provided a different set of quantitative signatures of metabolites that can be used to characterize the molecular changes in human urine samples. A comparison of normalized metabonomics data for selected metabolites in human urine samples in the presence of potential overlapping peaks after tea ingestion from LC/MS and (1)H NMR showed the reliability of the current approach and method of normalization. The close agreements of LC/MS with (1)H NMR data showed that the effects of ion suppression in LC/MS for early eluting metabolites were not significant. Concurrently, the specificity of detecting the stated metabolites by (1)H NMR and LC/MS was demonstrated. Our data showed that a number of metabolites involved in glucose metabolism, citric acid cycle and amino acid metabolism were affected immediately after the intake of green tea. The proposed approach provided a more comprehensive picture of the metabolic changes after intake of green tea in human urine. The multiple analytical approach together with pattern recognition tools is a useful platform to study metabolic profiles after ingestion of botanicals and medicinal plants.     

Use of liquid chromatography/time-of-flight mass spectrometry and multivariate statistical analysis shows promise for the detection of drug metabolites in biological fluids

The process of metabolite identification is essential to the drug discovery and development process; this is usually achieved by liquid chromatography/tandem mass spectrometry (LC/MS/MS) or a combination of liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) spectroscopy. Metabolite identification is, however, a time-consuming process requiring an experienced skilled scientist. Multivariate statistical analysis has been used in the field of metabonomics to elucidate differences in endogenous biological profiling due to a toxic effect or a disease state. In this paper we show how a combination of liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) and multivariate statistical analysis can be used to detect drug metabolites in a biological fluid with no prior knowledge of the compound administered.     

基于花生四烯酸代谢通路研究水飞蓟素对脂多糖诱导炎症模型影响

以脂多糖类似物(KLA)诱导的RAW264.7细胞为研究对象,采用代谢组学研究手段,研究水飞蓟素对脂多糖诱导炎症模型中花生四烯酸代谢通路的影响。以超高效液相色谱-三重四极杆质谱联用为平台,对不同浓度水飞蓟素作用下KLA诱导RAW264.7炎症细胞分泌的类二十烷酸代谢物进行定量分析,通过考察主成分分析(PCA)、正交偏最小二乘法判别分析(OPLS-DA)的VIP值和Kruskal-Wallis秩和检验结果显著性差异(P)值筛选代谢标记物。建立了59种类二十烷酸(含15种同位素内标)在5 min内实现快速分离的液相色谱-质谱联用方法;确定了细胞存活率在58%~80%的水飞蓟素浓度为50~150μmol/L;筛选出数据处理结果同时满足变异权重参数(VIP)值1且结果P值0.05的类二十烷酸代谢标记物12-OxoLeukotriene B4(12-OxoLTB4);通过分析柱状图和炎症信号通路,确定水飞蓟素借助其抗氧自由基特性发挥抗炎作用,通过抑制脂氧合酶-5(5-LOX)的活性及阻断5-LOX代谢通路中产生氧自由基的脂质过氧化反应来减少氧自由基及过氧化物的形成。综上所述,所建立的方法能快速准确地定量分析多种类二十烷酸,并从代谢组学角度解释了水飞蓟素的抗炎机制。     

Metabonomics Study of TCM Formula: Qutan Huayu Tongmai Granule as an Effective Treatment for Atherosclerosis in Mini-Pigs

To investigate the effects of QuTan HuaYu TongMai granule (QHTG) on the perturbed metabolism of atherosclerosis mini-pig model, a serum metabonomics method based on Liquid chromatography/mass spectrometry was developed. Principal component analysis and partial least squares-discriminate analysis models were established for the metabonomics analysis. The effect of high dose QHTG group is equivalent with those of positive drugs group in PCA score plots. Some significantly changed metabolites, including lyso-PCs, metabolites of vitamin D₃ and fatty acids were found to be reasonable in explaining the anti-atherosclerosis mechanism of QHTG. Metabonomic approach is helpful to further understand the pathophysiology of atherosclerosis in mini-pigs and the therapeutic mechanism of QHTG. Our work also indicated that the LC–MS based metabonomics method is a potential tool for performing intervention and mechanism research of traditional Chinese medicine (TCM).

     

Metabonomics Study of Heart Homogenates from Myocardial Infarction Rats Using Liquid Chromatography/Time of Flight Mass Spectrometry

In this study, a metabonomics analysis of heart homogenates from myocardial ischemic rats was performed by LC–TOF–MS. Hydrophilic interaction chromatography (HILIC) was used to separate the endogenous metabolites in heart homogenates. Partial least squares to latent structure-discriminant analysis (PLS-DA) was used for data analysis. Good separations were observed between the normal and model groups and 15 potential biomarkers were identified. The major disturbed metabolic pathways were purine metabolism, pyrimidine metabolism, urea cycle, and energy metabolism. The results demonstrated that a metabonomics approach based on HILIC-MS was useful for studying metabolic mechanism on target tissue of the myocardial infarction rat.

     

General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: Applications and constraints

LC coupled to single (LC–MS) and tandem (LC–MS/MS) mass spectrometry is recognized as the most powerful analytical tools for metabolic studies in drug discovery. In this article, we describe five cases illustrating the utility of screening xenobiotic metabolites in routine analysis of forensic samples using LC–MS/MS. Analyses were performed using a previously published LC–MS/MS general unknown screening (GUS) procedure developed using a hybrid linear IT–tandem mass spectrometer. In each of the cases presented, the presence of metabolites of xenobiotics was suspected after analyzing urine samples. In two cases, the parent drug was also detected and the metabolites were merely useful to confirm drug intake, but in three other cases, metabolite detection was of actual forensic interest. The presented results indicate that: (i) the GUS procedure developed is useful to detect a large variety of drug metabolites, which would have been hardly detected using targeted methods in the context of clinical or forensic toxicology; (ii) metabolite structure can generally be inferred from their “enhanced” product ion scan spectra; and (iii) structure confirmation can be achieved through in vitro metabolic experiments or through the analysis of urine samples from individuals taking the parent drug.     

Structure elucidation of three isomeric metabolites of SYN-2836, a novel antifungal agent, in dogs via liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry methodologies.

This paper presents liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) approaches for the rapid characterization of three urinary isomeric metabolites and their two precursor metabolites of SYN-2836, a novel antifungal agent, in dogs administered multiple oral doses of the agent (30 mg kg(-1) day(-1)). A collection of correlative data regarding the SYN-2836 metabolites was obtained by LC/MS and LC/MS/MS performed under complementary conditions such as the columns (C(18) vs cyano type), the mobile phase systems (acetonitrile-water-formic acid vs acetonitrile-water-ammonium acetate) and the electrospray ionization modes (positive vs negative). Metabolite identification was accomplished based on not only the LC/MS/MS data (product ion spectra) but also the LC/MS data indicating chromatographic behaviors of the metabolites. SYN-2836 and SYN-2869, an analog of the former, showed almost the same metabolic pathways following the same multiple-dose administration of the individual agents to the dogs. Therefore, correlation analysis in product ion spectra between corresponding metabolites of SYN-2836 and SYN-2869, and also in metabolic pathways between the two agents, was strategically used to facilitate the identification of the SYN-2836 (and SYN-2869 if necessary) metabolites. For the reason that various elucidation strategies were used complementarily, the chemical structures of the metabolites were unambiguously attained and the isomeric metabolites were explicitly differentiated without the use of other analytical methods. The methodologies used in this study may be applicable to metabolite screening of several structurally related agents simultaneously, promoting lead finding and optimization of drug candidates using a metabolism-based approach.     

Strategies for ascertaining the interference of phase II metabolites co‐eluting with parent compounds using LC–MS/MS

LC‐MS/MS is currently the most selective and efficient tool for the quantitative analysis of drugs and metabolites in the pharmaceutical industry and in clinical assays. However, phase II metabolites sometimes negatively affect the selectivity and efficiency of the LC‐MS/MS method, especially for the metabolites that possess similar physicochemical characteristics and generate the same precursor ions as their parent compounds due to the in‐source collision‐induced dissociation during the ionization process. This paper proposes some strategies for examining co‐eluting metabolites existing in real samples, and further assuring whether these metabolites could affect the selectivity and accuracy of the analytical methods. Strategies using precursor‐ion scans and product‐ion scans were applied in this study. An example drug, namely, caffeic acid phenethyl ester, which can generate many endogenous phase II metabolites, was selected to conduct this work. These metabolites, generated during the in vivo metabolic processes, can be in‐source‐dissociated to the precursor ions of their parent compounds. If these metabolites are not separated from their parent compounds, the quantification of the target analytes (parent compounds) would be influenced. Some metabolites were eluted closely to caffeic acid phenethyl ester on LC columns, although long columns and relatively long elution programs were used. The strategies can be utilized in quantitative methodologies that apply LC‐MS/MS to assure the performance of selectivity, thus enhancing the reliability of the experimental data.     

基于液相色谱-质谱联用技术的代谢组学方法在细胞种属分类中的应用

细胞内的代谢产物可以反映细胞的生理状态。为了考察基于胞内代谢物的指纹图谱对不同种属细胞进行区分的可行性,利用基于超高效液相色谱-高分辨飞行时间质谱(UPLC-TOF MS)技术的代谢组学方法对5种不同来源的细胞进行分类,获得了小分子代谢产物的差异表达谱,并采用主成分分析(PCA)数据处理方法对各类细胞进行模式识别。研究结果表明,不同的细胞种属之间均能呈现显著性差异。该研究可从分子水平对细胞种属进行分类,为细胞种属的鉴定与评价提供了一种新的技术方法,为细胞组学的深入研究提供了一种潜在的、非常具有应用前景的技术手段。