植物代谢组学中几种重要的次生代谢物液质分析技术研究进展

代谢组学（metabolomics）主要是研究生物体、组织、细胞的代谢物组分及检测其动态变化过程,是继基因组和蛋白组学后新兴的一门组学技术。代谢物是细胞调节过程中的最终产物,其水平被视为生物系统对遗传或环境变化的最终反映。通过合适的分析平台,准确定性、定量在复杂的生物中具有化学多样性的次生代谢物是代谢组学的一项重要工作。液相色谱-串联质谱技术（liquid chromatography-tandem mass spectrometry,LC-MS/MS）是代谢物质检测平台最常用的方法,也为植物次生代谢物的广泛应用研究提供了基础。本文主要从植物激素类、叶酸类、黄酮类等次生代谢物方面进行阐述,结合液质联用技术,简要论述不同次生代谢物检测技术的研究进展。     

基于超高效液相色谱-串联质谱技术的柑橘黑点病菌(Diaporthe citri)发育关联代谢物分析

【目的】柑橘黑点病是柑橘间座壳菌(Diaporthe citri)引起的真菌性病害,是危害柑橘的重要病害之一,D. citri在生长发育过程中经历菌丝生长(10 d, T1)、分生孢器形成(20 d, T2)和分生孢器产孢(30 d, T3)三个阶段。通过不同发育阶段代谢组分析,挖掘病原菌发育过程中标记物、关键代谢物,为黑点病菌产孢机制、代谢调控等深入研究提供依据。【方法】利用超高效液相色谱-串联质谱(ultra performance liquid chromatography/tandem mass spectrometry, UPLC-MS/MS)技术分析了D. citri发育过程中的代谢变化,采用主成分分析(principal component analysis, PCA)和正交偏最小二乘判别分析(orthogonal partial least squares discriminant analysis, OPLS-DA),筛选出了显著差异代谢物并进行了KEGG (Kyoto encyclopedia of genes and genomes)富集分析。【结果】D. cit...     

A well‐characterised peak identification list of MALDI MS profile peaks for human blood serum

MALDI MS profiling, using easily available body fluids such as blood serum, has attracted considerable interest for its potential in clinical applications. Despite the numerous reports on MALDI MS profiling of human serum, there is only scarce information on the identity of the species making up these profiles, particularly in the mass range of larger peptides. Here, we provide a list of more than 90 entries of MALDI MS profile peak identities up to 10 kDa obtained from human blood serum. Various modifications such as phosphorylation were detected among the peptide identifications. The overlap with the few other MALDI MS peak lists published so far was found to be limited and hence our list significantly extends the number of identified peaks commonly found in MALDI MS profiling of human blood serum.     

Influence of plant secondary metabolites on in vitro oxidation of methyl ferulate with cell wall peroxidases from lupine apoplast

Ionically bound cell wall peroxidases (POXs) were liberated to intercellular washing fluids (IWFs) and isolated together with other proteins and metabolites present in the apoplast of white lupine (Lupinus albus L. var. Bac) root. After separation of proteins from low molecular weight compounds, activity of peroxidases was monitored in in vitro experiments. Oxidation of methyl ferulate with H2O2 was studied in multi-component mixtures of plant metabolites. Secondary metabolites identified in IWFs or other natural products playing important roles in different physiological processes were applied as modifiers of the dehydrodimerization process during oxidation reactions performed in vitro. These were isoflavones and their conjugates, lupanine representing quinolizidine alkaloids synthesized in lupine, or other natural products such as quercetin, ascorbic, and salicylic acid. The influence of these substances on the oxidation kinetics of methyl ferulate was monitored with liquid chromatography with ultraviolet detection (LC/UV), and identification of compounds was confirmed with the liquid chromatography/mass spectroscopy (LC/MS) system. On the basis of data collected, it was possible to reveal changes in the activities of cell wall POXs. Application of the LC system permitted us to monitor, independently, quantitative changes of two or more reaction products in the mixtures. In multi-component combinations, oxidation yields of methyl ferulate by POXs were modified depending on the actual composition of the reaction mixture. We conclude that various classes of plant secondary metabolites can modify the yield of methyl ferulate oxidation by hydrogen peroxide in the presence of POX, due to interactions with the enzyme's active site (genistein) or radical scavenging properties of metabolites present in the reaction mixture.     

Profiling of phenolic glycosidic conjugates in leaves of Arabidopsis thaliana using LC/MS

Profiling of plant secondary metabolites is still a very difficult task. Liquid chromatography (LC) or capillary electrophoresis hyphenated with different kinds of detectors are methods of choice for analysis of polar, thermo labile compounds with high molecular masses. We demonstrate the applicability of LC combined with UV diode array or/and mass spectrometric detectors for the unambiguous identification and quantification of flavonoid conjugates isolated from Arabidopsis thaliana leaves of different genotypes and grown in different environmental conditions. During LC/UV/MS/MS analyses we were able to identify tetra-, tri-, and di-glycosides of kaempferol, quercetin and isorhamnetin. Based on our results we can conclude that due to the co-elution of different chemical compounds in reversed phase HPLC systems the application of UV detectors does not allow to precisely profile all flavonoid conjugates existing in A. thaliana genotypes. Using MS detection it was possible to unambiguously recognize the glycosylation patterns of the aglycones. However, from the mass spectra we could not conclude neither the anomeric form of the C-1 carbon atoms of sugar moieties in glycosidic bonds between sugars or sugar and aglycone nor the position of the second carbon involved in disaccharides. The applicability of collision induced dissociation techniques (CID MS/MS) for structural analyses of the studied group of plant secondary metabolites with two types of analyzers (triple quadrupole or ion trap) was demonstrated.     

Factors affecting the production of Trichoderma harzianum secondary metabolites during the interaction with different plant pathogens

Aims: Strains of Trichoderma spp. produce numerous bioactive secondary metabolites. The in vitro production and antibiotic activities of the major compounds synthesized by Trichoderma harzianum strains T22 and T39 against Leptosphaeria maculans, Phytophthora cinnamomi and Botrytis cinerea were evaluated. Moreover, the eliciting effect of viable or nonviable biomasses of Rhizoctonia solani, Pythium ultimum or B. cinerea on the in vitro production of these metabolites was also investigated. Methods and Results: T22azaphilone, 1‐hydroxy‐3‐methyl‐anthraquinone, 1,8‐dihydroxy‐3‐methyl‐anthraquinone, T39butenolide, harzianolide, harzianopyridone were purified, characterized and used as standards. In antifungal assays, T22azaphilone and harzianopyridone inhibited the growth of the pathogens tested even at low doses (1–10 μg per plug), while high concentrations of T39butenolide and harzianolide were needed (>100 μg per plug) for inhibition. The in vitro accumulation of these metabolites was quantified by LC/MS. T22azaphilone production was not enhanced by the presence of the tested pathogens, despite its antibiotic activity. On the other hand, the anthraquinones, which showed no pathogen inhibition, were stimulated by the presence of P. ultimum. The production of T39butenolide was significantly enhanced by co‐cultivation with R. solani or B. cinerea. Similarly, viable and nonviable biomasses of R. solani or B. cinerea increased the accumulation of harzianopyridone. Finally, harzianolide was not detected in any of the interactions examined. Conclusions: The secondary metabolites analysed in this study showed different levels of antibiotic activity. Their production in vitro varied in relation to: (i) the specific compound; (ii) the phytopathogen used for the elicitation; (iii) the viability of the elicitor; and (iv) the balance between elicited biosynthesis and biotransformation rates. Significance and Impact of the Study: The use of cultures of phytopathogens to enhance yields of Trichoderma metabolites could improve the production and application of novel biopesticides and biofertilizers based on the active compounds instead of the living microbe. This could have a significant beneficial impact on the management of diseases in crop plants.     

LC-MS/MS-based metabolic profiling of Escherichia coli under heterologous gene expression stress

Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin-resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)-induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography-mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN-based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA-CoA, p-aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l -carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules.     

昆虫对植物次生物质的代谢适应机制及其对昆虫抗药性的意义

植物次生物质(plant secondary metabolites)对昆虫的取食行为、生长发育及繁殖可以产生不利影响,甚至对昆虫可以产生毒杀作用。为了应对植物次生物质的不利影响,昆虫通过对植物次生物质忌避取食、解毒代谢等多种机制,而对寄主植物产生适应性。其中,昆虫的解毒代谢酶包括昆虫细胞色素P450酶系（P450s）及谷胱甘肽硫转移酶（GSTs）等,在昆虫对植物次生物质的解毒代谢及对寄主植物的适应性中发挥了重要作用。昆虫的解毒酶系统不仅可以代谢植物次生物质,还可能代谢化学杀虫剂,因而昆虫对寄主植物的适应性与其对杀虫剂的耐药性甚至抗药性密切相关。昆虫细胞色素P450s和GSTs等代谢解毒酶活性及相关基因的表达可以被植物次生物质影响,这不仅使昆虫对寄主植物的防御产生了适应性,还影响了昆虫对杀虫剂的解毒代谢,因而改变昆虫的耐药性或抗药性。掌握昆虫对植物次生物质的代谢适应机制及其在昆虫抗药性中的作用,对于明确昆虫的抗药性机制具有重要的参考意义。本文综述了植物次生物质对昆虫的影响、昆虫对寄主植物次生物质的代谢机制、昆虫对植物次生物质的代谢适应性对昆虫耐药性及抗药性的影响等方面的研究进展。     

Simultaneous LC-MS/MS Analysis of Glyphosate,Glufosinate, and Their Metabolic Products in Beer,Barley Tea,and Their Ingredients

Glyphosate and glufosinate are non-selective herbicides that have been extensively used worldwide. Their ionic and water-soluble characteristics often make it difficult to analyze them, especially in food components. A method was developed in this study for the simultaneous analysis of glyphosate, glufosinate, and three metabolic products in beer, barley tea, and their ingredients (malt and corn). The analytical samples were extracted with H₂O, purified with a strong anion-exchange solid-phase extraction (SPE) cartridge, and then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with an anion-exchange high-performance liquid chromatography (HPLC) column. This method enabled a rapid and sensitive analysis [limit of quantification (LOQ) = 10 µg/kg] of the herbicides to be achieved.     

表观遗传的化学干预对金龟子绿僵菌次生代谢产物影响的研究

[背景] 表观遗传酶类化学抑制剂对真菌的影响研究主要集中在新次生代谢产物挖掘方面,而对大量已知次生代谢物含量的变化却关注较少。金龟子绿僵菌是一种常用杀虫真菌,能代谢出多种已知生物活性物质,其含量可能会影响到该菌与环境间关系及利用潜力。[目的] 评估组蛋白去乙酰化酶和DNA甲基转移酶的化学抑制剂对金龟子绿僵菌代谢物安全性和可利用性的影响。[方法] 在金龟子绿僵菌培养基中添加表观遗传酶类化学抑制剂,培养一定时间后用高分辨液质联用及标准品对照方法分析次生代谢产物变化。根据差异代谢物的生物活性评估化学抑制剂的影响。[结果] 高分辨液质联用分析结果表明当抑制剂浓度达500μmol/L时,金龟子绿僵菌有16种主要次生代谢产物出现明显量的变化,包括destruxin A、A1、A2、B、B1、B2、E、E2、Ed、didesmethyldestruxin C、dihydrodestruxin A、desmethyldestruxin B、12-hydroxyovalicin、subglutinol C、fungerin和ustilagic Acid C。其中,丁酸钠处理可使15种主要代谢物含量升高。苯甲酰胺可使12种主要代谢物含量升高。伏立诺他虽然仅能使10种主要代谢物含量升高,但部分代谢物的升高幅度明显高于前两者。2种DNA甲基转移酶抑制剂可使金龟子绿僵菌代谢物中绿僵菌素类代谢物含量普遍下降。[结论] 组蛋白去乙酰化酶抑制剂可引起金龟子绿僵菌主要代谢物含量普遍升高,而DNA甲基转移酶抑制剂使金龟子绿僵菌的绿僵菌素含量普遍下降。由于变化的代谢物都具有显著的杀虫、免疫抑制或抗菌抗癌等生物活性,因此上述化学抑制剂可增强或降低金龟子绿僵菌对环境中昆虫毒性,同时也增加或降低其代谢物利用潜力。另外,subglutinol C、fungerin和ustilagic Acid C是首次在金龟子绿僵菌中被发现。     

基于GC-MS和UPLC-QTOF/MS技术的灵芝孢子粉化学成分分析

采用极性不同的6种溶剂（石油醚、乙酸乙酯、丙酮、乙醇、甲醇和水）、按索氏提取法逐级萃取破壁灵芝孢子粉,并同时运用气相色谱-质谱联用（GC/MS）和超高效液相串联四极杆飞行时间质谱（UPLC-QTOF/MS）技术对各萃取物进行化学成分分析与鉴定。结果表明：GC/MS共鉴定出101种化合物,其中酸类10种、酯类40种、醇类7种、酮类6种、酚类2种、烃类18种、甾类9种和杂原子化合物9种;UPLC-Q-TOF/MS共推断出40种化合物,其中倍半萜类1种、二萜类1种、三萜类9种、生物碱类4种、酰胺类7种、有机酸类9种以及其他化合物9种。两种测定方法间共有化合物仅1种,仅存在于5种有机溶剂（石油醚、乙酸乙酯、丙酮、乙醇和甲醇）萃取物之一的化合物共105种,2种或2种以上萃取物共有的化合物共31种,实验方法较好地实现了样品中化合物组分的充分分离,扩大了可检测化合物的范围。研究结果为灵芝孢子粉中化学成分的系统分析与鉴定、及灵芝孢子粉的化合物谱图库的完善提供了基础资料,为相关药理、药效分析及灵芝的药用模式真菌研究提供参考。     

6种植物次生物质对斜纹夜蛾解毒酶活性的影响

草食性昆虫取食植物时遇到宿主植物中大量次生物质的化学防御,研究昆虫适应植物毒素的反防御策略具有重要的科学意义。分别添加0.01%肉桂酸、0.01%水杨酸、0.01%花椒毒素、0.02%槲皮素、0.05%黄酮和0.1%香豆素等6种植物次生物质的人工饲料饲养斜纹夜蛾(Spodoptera litura)五龄幼虫48 h后,测定斜纹夜蛾幼虫中肠和脂肪体中谷胱甘肽S-转移酶(GSTs)、羧酸酯酶(CarE)、P450的酶含量及头部乙酰胆碱酯酶(AChE)的活性,利用半定量RT-PCR检测中肠和脂肪体中CYP4M14和CYP4S9的基因表达水平。结果表明:取食肉桂酸和香豆素后,斜纹夜蛾中肠中CarE的酶活性分别提高了1.67和1.37倍,取食6种次生物质均能显著提高斜纹夜蛾脂肪体中GSTs酶活性。取食肉桂酸和香豆素48 h后,脂肪体中P450酶含量比对照增加2.93和14.50倍。取食肉桂酸、花椒毒素、槲皮素和香豆素后,斜纹夜蛾头部AchE酶活性与对照相比提高了1.53、1.80、2.36和1.56倍。6种次生物质均可诱导脂肪体中CYP4M14基因表达,槲皮素、肉桂酸和香豆素强烈诱导CYP4S9在脂肪体中表达。表明,斜纹夜蛾具有利用植物次生物质诱导其解毒酶的能力,进而提高其对毒素的抗性。     

Determination of volatile products of human colon cell line metabolism by GC/MS analysis

Colon cancer is one of the most reasons for cancer death worldwide. Thus, it is important to find new prognostic and diagnostic marker, as well as to throw light on the special metabolic pathways of colon cancer cells. This paper highlights for the first time some qualitative differences in the profiles of the volatile metabolites of colon cancer cell lines SW 480 (grade IV, Duke B) and SW 1116 (grade II, Duke A) among themselves and in comparison to the normal colon cell line NCM460, which are mostly represented by ketones and alcohols. These results, which were obtained by applying solid phase micro extraction (SPME) and combined gas chromatography/mass spectrometry (GC/MS), are consistent with Warburg’s hypothesis because the found reaction products may indicate that the cancer cells show the Crabtree’s effect. Furthermore, compounds like undecan-2-ol and pentadecan-2-one were associated for the first time with the human metabolism. In summary, these findings indicate that the metabolism of colon cancer cells differs extremely from the metabolism of healthy cells and it changes during the progress of the disease. Compounds that are present in the breath, the blood and the tissue of patients represent the differences and they can serve as new biomarker for colon cancer in future.     

MMS2plot: An R Package for Visualizing Multiple MS/MS Spectra for Groups of Modified and Non‐Modified Peptides

A large number of post‐translational modifications (PTMs) in proteins are buried in the unassigned mass spectrometric (MS) spectra in shot‐gun proteomics datasets. Because the modified peptide fragments are low in abundance relative to the corresponding non‐modified versions, it is critical to develop tools that allow facile evaluation of assignment of PTMs based on the MS/MS spectra. Such tools will preferably have the ability to allow comparison of fragment ion spectra and retention time between the modified and unmodified peptide pairs or group. Herein, MMS2plot, an R package for visualizing peptide‐spectrum matches (PSMs) for multiple peptides, is described. MMS2plot features a batch mode and generates the output images in vector graphics file format that facilitate evaluation and publication of the PSM assignment. MMS2plot is expected to play an important role in PTM discovery from large‐scale proteomics datasets generated by liquid chromatography‐MS/MS. The MMS2plot package is freely available at https://github.com/lileir/MMS2plot under the GPL‐3 license.     

A simple and rapid GC/MS method for the simultaneous determination of gaseous metabolites

We modified and tuned a commercial model of a gas chromatography/mass spectrometry (GC/MS) instrument to develop a simple and rapid method for the simultaneous quantification of a variety of gas species. Using the developed method with the newly modified instrument, gas species such as H₂, N₂, O₂, CO, NO, CH₄, CO₂, and N₂O, which are common components of microbial metabolism, were accurately identified based on their retention times and/or mass-to-charge ratios (m/z) in less than 2.5 min. By examining the sensitivities and dynamic ranges for the detection of H₂, N₂, O₂, CH₄, CO₂, and N₂O, it was demonstrated that the method developed in this study was sufficient for accurately monitoring the production and the consumption of these gaseous species during microbial metabolism. The utility of the new method was demonstrated by a denitrification study with Pseudomonas aureofaciens ATCC 13985^T. This method will be suitable for a variety of applications requiring the identification of gaseous metabolites in microorganisms, microbial communities, and natural ecosystems.     

基于LC-MS/MS分析马缨杜鹃花代谢物的变化

为分析马缨杜鹃(Rhododendron delavayi)花开花至凋谢过程中的代谢产物差异及其通路,该文采用LC-MS/MS技术对其花苞期、开裂期、传粉期、盛开期、衰老期和凋谢期的化学成分进行非靶向代谢组学分析。结果表明:(1)共鉴定到973种代谢物,主要包含黄酮类、有机酸、酚酸类、氨基酸及其衍生物、脂类、生物碱等。(2)主成分分析(PCA)表明样本间代谢物存在差异,结合正交偏最小二乘判别分析(OPLS-DA)、t检验的P值和单变量分析的差异倍数(fold-change)筛选差异代谢物(VIP>1,P<0.05,Fc>2或Fc<0.5),涉及591种,在马缨杜鹃花期进入衰老期和凋谢期后差异代谢物数量和表达量显著上升,其中花苞期至开裂期差异代谢物的表达主要呈现下调,而进入衰老期和凋谢期后差异代谢物的表达主要呈现上调。(3)KEGG注释到68条代谢通路,其中差异代谢物极显著富集(P < 0.01)通路3条,包括苯丙素类生物合成、植物激素的生物合成和类黄酮生物合成。(4)结合苯丙素类、黄酮类等有效成分生物合成通路共筛选到10种代谢物包括苯丙氨酸(L-phenylalanine)、反式肉桂酸(trans-cinnamic acid)、查耳酮(chalcone)、柚皮素(naringenin)、对香豆酰基莽草酸(p-coumaroyl shikimic acid)、阿魏酸(ferulic acid)、松柏醇(coniferyl alcohol)、芥子酸(sinapic acid)、紫丁香苷(syringin)、槲皮素(quercetin)。此外,有效成分的差异代谢物表明苯丙素类生物合成代谢活动随马缨杜鹃花的发育逐渐增强,而黄酮类化合物生物合成逐渐减弱,这些关键差异代谢物可能对马缨杜鹃花的发育有重要的调控作用。该研究为马缨杜鹃花开花至凋谢进程中的有效成分代谢途径活性物质的研究提供了代谢组学基础,为进一步研究马缨杜鹃花花期调控的分子机理提供参考。     

穆棱林区野生东北马鹿种群冬季食物组成模式与植物代谢产物的关系

高纬度地带,在冬季食物资源有限的环境中,野生大型有蹄类动物满足营养需求的同时,需要对植物中次生代谢产物进行平衡,回避有害物质并选择对机体有益的成分,从而形成特定的食物组成模式。以东北马鹿（Cervus elaphus xanthopygus）为研究对象,于2020年11月,在黑龙江穆棱东北红豆杉国家级自然保护区境内,采集东北马鹿粪便和植物样本。通过粪便显微分析法确定保护区内马鹿冬季食物组成,采用k-means聚类分析揭示马鹿冬季食物组成模式。应用广泛靶向代谢组技术对部分植物中次生代谢产物的含量进行全覆盖定性和相对定量检测,将食物组成与次生代谢产物数据整合,进行曼特尔检验（Mantel test）分析,以探究植物次生代谢产物对马鹿种群内食物组成模式的影响。结果表明,林区内马鹿种群冬季共采食30种植物,其中木本植物占92.48%;且种群内分别呈现出以东北红豆杉（Taxus cuspidata）,簇毛槭（Acer barbinerve）,毛榛子（Corylus mandshurica）为主要食物的不同食物组成模式。广泛靶向代谢组技术共检测出638种次生代谢产物,有25种代谢物与马鹿采食频率显著相关,其中多数萜类物质抑制马鹿采食,而鞣质类物质对马鹿的采食选择有一定的正向作用;Mantel test结果显示,上述25种物质中黄酮、鞣质、萜类化合物相对含量与不同马鹿个体食物组成显著相关,说明这些代谢物相对含量和性质的差异会造成种群内不同个体食物组成的差异,是种群内形成不同食物组成模式的原因之一。从植物次生代谢产物角度揭示了该地区东北马鹿种群冬季食物组成模式呈现差异的可能因素,为进一步研究大型有蹄类营养策略和植物化学防御关系提供基础依据。