[Show abstract][Hide abstract] ABSTRACT: Abiotic stress caused by insecticide treatment is an interesting and challenging topic in plant research. Here a simultaneous extraction method with methyl tert-butyl ether for metabolomics and lipidomics analysis by using rapid resolution liquid chromatography quadrupole time-of-flight mass spectrometry was developed and validated. The extraction efficiency of lipidome and metabolome based on the current developed method showed an obvious improvement compared with literatures. About 300 metabolites were identified in rice leaf. Method validation was performed and analytical properties including the linearity (R2, 0.991–1.000), repeatability (over 87 % peaks with CV cry1Ac and sck genes and its wild counterpart. Antioxidants including ferulic acid and sinapic acid were down-regulated at 24 h after insecticide treatment. Signaling metabolites including salicylic acid and nicotinamide adenine dinucleotide were significantly up-regulated at 12–24 h after treatment in transgenic rice. More flavonoids were significantly changed in transgenic plants. Results indicated that gene transformation affected the metabolic response of rice to insecticide stress.
[Show abstract][Hide abstract] ABSTRACT: A pseudotargeted method based on gas chromatography and mass spectrometry with selected-ion monitoring method was established to investigate the metabolite differences of flue-cured tobacco from three different growing regions. The mixed solvent of acetonitrile/isopropanol/water (3:3:2, v/v/v) was chosen as the optimal extraction system based on the good repeatability and extraction efficiency. A self-developed software coupled with commercial software was used to establish the pseudotargeted method including 289 peaks and 47 groups. Multivariable statistical analysis indicated that tobacco samples can be obviously separated based on the geographical origins. On the basis of a Mann-Whitney U test, organic acids, phenols and alkaloids had higher levels in Hunan province. In contrast, a large proportion of amino acids (including l-tyrosine, l-proline, and serine), sucrose and linoleic acid were the highest in Yunnan province. Meanwhile, multiple metabolic pathways (including carbohydrate metabolism, tricarboxylic acid cycle, and nitrogen metabolism) were influenced by growing regions. Twenty-eight differential metabolites, which had great contributions to the classification of tobacco samples of three growing regions, were further defined. The results demonstrated that the developed pseudotargeted method was a powerful tool to investigate the metabolic profiling of tobacco leaves and discriminate tobacco leaves of different growing regions. This article is protected by copyright. All rights reserved.
Journal of Separation Science 05/2014; · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cured tobacco is an important plant material. Component studies are a big challenge for its significantly diverse chemical properties and vastly different concentrations. In this work, liquid chromatography with quadrupole time-of-flight mass spectrometry was used to perform a metabolomics study of cured tobacco owing to its efficient separation and detection of semi-polar metabolites. A solvent of methanol/water (8:2, v/v) and 30 min of ultrasound time were found to be optimal to perform extraction. 95, 92 and 93% of metabolite features had within 20% of coefficient of variation for repeatability, intra-day and inter-day precision analysis, respectively, indicating a good stability of the method developed. 113 metabolites were identified in cured tobacco based on accurate mass, retention time and MS/MS fragments. The developed method was applied to a metabolomics study of cured tobacco from three growing regions. 43 metabolites were found to be contributed to the classification. It is shown that the developed method can be applied to metabolomics analysis of plant materials. This article is protected by copyright. All rights reserved.
Journal of Separation Science 02/2014; · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many metabolites in plant are highly polar and ionic. Their analysis using gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry can be problematic. Therefore a capillary electrophoresis–mass spectrometry (CE–MS) method with charge-driven separation characteristic was developed to investigate polar metabolites in tobacco. To obtain as many features as possible, extraction of polar metabolites was optimized by the design of experiments and evaluated by univariate statistics. Method validation was carried out to evaluate the analytical characteristics including calibration curve, precision, sample stability and extraction reproducibility. The developed method was successfully applied in studying 30 tobacco leaves obtained from Yunnan and Guizhou provinces in China. A total of 154 polar metabolites were identified based on available database. Multivariate pattern recognition clearly revealed the metabolic differences between the two geographic areas and 43 significantly different metabolites were defined by the non-parametric hypothesis test (Mann–Whitney U test) and false discovery rate. Some key metabolites involved in photosynthesis such as ribulose 1,5-disphosphate, fructose 1,6-diphosphate, glycine, betaine, GABA and serine were found to be susceptible to environmental conditions. This study shows that the metabolic profiling based on CE–MS can clearly discriminate tobacco leaves of different geographical origins and understand the relationship between plant metabolites and their geographical origins.
[Show abstract][Hide abstract] ABSTRACT: Twenty-five phenolic compounds in tobacco were analyzed using high performance liquid chromatography-ultraviolet/mass spectrometry (HPLC-UV/MS), including absolute quantification of 11 main phenols by HPLC-UV and relative quantification of other 14 phenols by SHPLC-MS. The validation results of this method were satisfactory. Eleven phenol standards showed good linearity with correlation coefficients (r2) of 0.999 3 - 0.999 9 over the mass concentration range from 0.90 mg/L to 99.00 mg/L (the range of chlorogenic acid and rutin was from 0.95 mg/L to 380.00 mg/L). The recoveries of the 11 phenol standards were 91.0% - 112.4% with the RSDs of 0.33% - 8.11% at the three spiked levels of 22.5 - 24.8, 45.0 - 49.5 and 67.5 - 74.3 mg/L. The reproducibility of the method was good with the RSDs of 1.48% - 13.40%. In addition, the intra-day and inter-day precisions were also satisfactory with the RSDs of 0.35% - 15.54%. Mature fresh tobacco leaves from Yunnan, Henan and Guizhou Provinces, China were analyzed using this method. The results showed that the total amount of main phenols was Guizhou > Yunnan > Henan and there was significant difference between Guizhou and Henan. The amount of rutin was Yunnan > Guizhou > Henan, and there were significant differences among them each other. The method is reproducible with a simple pretreatment covering a wide range of phenols, and can be applied in batch analysis of tobacco leaves.
Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 01/2014; 32(1):40-6.
[Show abstract][Hide abstract] ABSTRACT: An improved pseudo-targeted method using gas chromatography/mass spectrometry (GC/MS) was developed to investigate the metabolic profile of tobacco leaves from three planting regions (Yunnan, Guizhou and Henan provinces). The analytical characteristics of the method with regard to reproducibility, precision, linearity and stability were satisfactory for metabolic profiling study. Partial least squares-discriminant analysis and hierarchical cluster analysis demonstrated that the metabolic profiles of tobacco from the Yunnan and Guizhou regions were different from that from the Henan province. The amino acid (e.g., phenylalanine, leucine and tyrosine) and carbohydrate (e.g., fructose, trehalose and sucrose) contents were the highest in Henan tobacco. The highest contents of organic acids (e.g., isocitrate, citrate and fumarate) of the TCA cycle and antioxidants (e.g., quinate, chlorogenic acid and ascorbate) were found in Guizhou tobacco. The correlation coefficients between metabolite content and climate factors (rainfall, sunshine and temperature) demonstrated that drought facilitated the accumulation of sugars and amino acids. The content of TCA cycle intermediates could be influenced by multiple climate factors. This study demonstrates that the pseudo-targeted method with GC/MS is suitable for the investigation of the metabolic profiling of tobacco leaves and the assessment of differential metabolite levels related to the growing regions.
Journal of Proteome Research 10/2013; · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amino acids are one of the most important metabolites of organisms. They play an important role in plant growth, development and product quality. A method based on RP ultra-performance liquid chromatography with single quadrupole mass spectrometry and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate pre-column derivatization was developed for the analysis of free amino acids in flue-cured tobacco leaves. Different from the corresponding UV detection method, this method avoids matrix interference of complicated tobacco components, and the quantitative accuracy and resolution were improved. Twenty free amino acids were detected in flue-cured tobacco leaves. The method showed a good linearity with correlation coefficients of 0.9966-0.9998. The limits of detection for derivatized amino acids were 0.2-9.7 fmol/μL. Good repeatability with a RSD of 2.5-8.6% and satisfactory intra-day and inter-day precisions were obtained. The developed method was used to investigate free amino acids in flue-cured tobacco leaves in China. The effects of aroma type, variety and growing regions on free amino acids were investigated. The results showed that free amino acids in tobacco were affected by growing regions and varieties. This article is protected by copyright. All rights reserved.
Journal of Separation Science 06/2013; · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An analytical strategy for the metabolic profiling of rice grain was developed based on gas chromatography-mass spectrometry (GC-MS). For the purpose of obtaining abundant metabolite information, sample preparation step prior to instrumental analysis is necessary to be optimized. D-optimal experimental design was applied to optimize the extraction solvent. Four solvents, including water, methanol, isopropanol and acetonitrile, and their combinations were evaluated for the extraction efficiency using multivariate statistical analysis (partial least square regression). The count of resolved peaks and the sum of peak areas were taken as the evaluation indexes. Methanol/water (80:20, v/v) mixture was highly efficient for rice metabolites and was selected as the suitable solvent formulation. Then, the analytical characteristics of the method were measured. More than 90% of the metabolites had satisfactory precisions, reproducibilities and stabilities (relative standard deviations (RSDs) < 30%). Most of the detected metabolites (about 88.0% of total peak area) showed good linear responses. With the optimized analytical protocol, 315 metabolites were detected in rice and 86 of which were structurally identified by searching in the NIST 08/Wiley standard mass spectral library, covering carbohydrates, amino acids, organic acids, steroids and so on which showed a broad coverage of metabolite data. The established method is expected to be useful for the metabolomic studies of rice.
Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 10/2012; 30(10):1037-42.
[Show abstract][Hide abstract] ABSTRACT: In this work a chip-based nano HPLC coupled MS (HPLC-chip/MS) method with a simple sample preparation procedure was developed for the flavonoid profiling of soybean. The analytical properties of the method including the linearity (R(2) , 0.992-0.995), reproducibility (RSD, 1.50-7.66%), intraday precision (RSD, 1.41-5.14%) and interday precision (RSD, 2.76-16.90%) were satisfactory. Compared with the conventional HPLC/MS method, a fast extraction and analysis procedure was applied and more flavonoids were detected in a single run. Additionally, 13 flavonoids in soybean seed were identified for the first time. The method was then applied to the profiling of six varieties of soybean sowed at the same place. A clear discrimination was observed among different cultivars, three isoflavones, accounting for nearly 80% of total flavonoid contents, were found increased in the spring soybeans compared with the summer cultivars.
[Show abstract][Hide abstract] ABSTRACT: Insecticide is always used to control the damage from pests, while the potential influence on plants is rarely known. Time-course metabolic changes of wild and Bacillus thuringiensis (Bt) transgenic rice (Oryza sativa L.) plants after insecticide treatment were investigated by using gas chromatography-mass spectrometry (GC-MS). A combined statistical strategy of 2-way ANOVA and multivariate analyses (principal component analysis and hierarchal cluster analysis) was performed to find the stress-associated effects. The results reveal that a wide range of metabolites were dynamically varied in both varieties as a response to insecticide, in multiple metabolic pathways, such as biosynthesis and metabolism of amino acids, carbohydrates, fatty acids, TCA cycle, and the shikimate/phenylpropanoid pathway, and most of the changes were correlated with the exposure time and dependent on the variety. A set of stress defenses were activated, including phytohormone signaling pathway, antioxidant defense system, shikimate-mediated secondary metabolism, and so on. In particular, insecticide led to much stronger regulations of signaling molecules (salicylate and the precursor of jasmonate) and antioxidants (α-tocopherol and dehydroascorbate/ascorbate) in Bt-transgenic variety at the early stage. Our results demonstrated that the Bt-transgenic rice had a more acute and drastic response to insecticide stress than its non-transgenic counterpart in antioxidant system and signaling regulation.
Journal of Proteome Research 07/2012; 11(8):4351-60. · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As a primary characteristic of substantial equivalence, the evaluation of unintended effects of genetically modified plants has been evolving into an important field of research. In this study, a metabolic profiling method for rice seeds was developed using rapid resolution liquid chromatography/quadrupole time-of-flight mass spectrometry. The analytical properties of the method, including the linearity, reproducibility, intra-day precision and inter-day precision, were investigated and were found to be satisfactory. The method was then applied to investigate the differences between transgenic rice and its native counterparts, in addition to the differences found between native rice with different sowing dates or locations. Global metabolic phenotype differences were visualized, and metabolites from different discriminated groups were discovered using multivariate data analysis. The results indicated that environmental factors played a greater role than gene modification for most metabolites, including tryptophan, 9,10,13-trihydroxyoctadec-11-enoic acid, and lysophosphatidylethanolamine 16:0. The concentrations of phytosphingosine, palmitic acid, 5-hydroxy-2-octadenoic acid and three other unidentified metabolites varied slightly due to gene modification.
[Show abstract][Hide abstract] ABSTRACT: The Bacillus thuringiensis δ-endotoxin and cowpea trypsin inhibitor genes have been introduced into the rice genome to improve its pest resistance via Agrobacterium-mediated transformation. A gas chromatography-mass spectrometry (GC–MS) based metabolic profiling method was employed to
determine the unpredictable metabolic changes resulting from the gene insertion and tissue culture separately. Descendants
of the same transformant were obtained from different breeding programs, including both the transgenic and null-segregant
progeny. The comparison of the transgenic and respective null-segregant plants enabled the evaluation of variations caused
by transgenes; also the null-segregant plants were compared with the wild-type control to identify the influence of tissue
culture. Based on the GC–MS metabolic profiles, the principal component analysis and significant differences determined by
Student’s t-test suggested that there were more metabolic changes from the tissue culture than those from the insertion of the transgenes.
By comparing different breeding programs, it was clear that the progeny which was developed after several generations of backcross
with the non-transformed rice as the recurrent parent, displayed fewer metabolic differences from the non-transformed parent.
A GC–MS based metabolic profiling study confirmed that backcrossing can help to reduce unwanted variations that occur during
[Show abstract][Hide abstract] ABSTRACT: Nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) has attracted increasing interest in virtue of high sensitivity, low sample consumption, and minimal matrix effect. In this work a HPLC-Chip/quadrupole time-of-flight (Q-TOF) MS device with a new ultra-high capacity small molecule chip (UHC-Chip) which features a 500 nL enrichment column and a 150 mm × 75 μm analytical column, was evaluated with a drug mixture covering a wide range of polarities. Excellent chromatographic precision with 0.1-0.5% RSD for retention time and 1.7-9.0% RSD for peak area, low limit of detection, good chip-to-chip reproducibility and linearity were obtained by using this UHC-Chip. Compared with the standard HPLC-Chip with 40 nL trapping column, the UHC-Chip showed higher enrichment capability and hence gave a higher response in signal detection. Additionally, 4-30 times increase in sensitivity was obtained compared with conventional LC/MS, which indicated that UHC-Chip/MS was a valuable tool for the quantitative analysis of low level impurities and degradation products in pharmaceuticals. Moreover, satisfactory results obtained from trace drug analysis of serum samples further proved its practicality and potential for use in drug testing and development.
Journal of Chromatography A 06/2011; 1218(23):3669-74. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An approach was developed for extracting and analyzing the chemical components of tobacco leaves based on solvent extraction and rapid & resolution liquid chromatography/quadrupole time-of-flight mass spectrometry analysis. Two solvents with different polarities were used to extract hydrophilic components and hydrophobic components, respectively, the combined analytical data can provide a "global" view of metabolites. Based on the evaluation of parallel samples, it was found that this approach provided good repeatability, accurate and reliable profiling data, and is suitable for the metabolomics study of tobacco leaves. In order to find the chemical component differences of tobacco leaves, 56 samples from Zimbabwe and China were analyzed using the developed method. The metabolite data were processed by multivariate statistic technique; an obvious group classification between Zimbabwe and China was observed, 14 significantly changed compounds were found, and 9 of them were identified.
Journal of Separation Science 01/2011; 34(2):119-26. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Systematic studies were performed on the biological perturbations in metabolic phenotype responding to protein-energy malnutrition through global metabolic profiling analysis, in combination with pattern recognition. The malnutrition rat model was established through five weeks of strict diet restriction, and the metabonome data obtained from gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were integrated to approximate the comprehensive metabolic signature. Principal component analysis and orthogonal projection to latent structure analysis were used for the classification of metabolic phenotypes and discovery of differentiating metabolites. The perturbations in the urine profiles of malnourished rats were marked by higher levels of creatine, threitol, pyroglutamic acid, gluconic acid and kynurenic acid, as well as decreased levels of succinic acid, cis-aconitic acid, citric acid, isocitric acid, threonic acid, trimethylglycine, N-methylnicotinic acid and uric acid. The alterations in these metabolites were associated with perturbations in energy metabolism, carbohydrate, amino acid, and fatty acid metabolism, purine metabolism, cofactor and vitamin metabolism, in response to protein and energy malnutrition. Our findings show the integration of GC-MS and LC-MS techniques for untargeted metabolic profiling analysis was promising for nutriology.