Yuwei Chang

Metabolic profiling based on LC/MS to evaluate unintended effects of transgenic rice with cry1Ac and sck genes

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.

Ultra-high capacity liquid chromatography chip/quadrupole time-of-flight mass spectrometry for pharmaceutical analysis

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.

Alteration of leaf metabolism in Bt-transgenic rice (Oryza sativa L.) and its wild type under insecticide stress.

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.

Investigation of the Relationship between the Metabolic Profile of Tobacco Leaves in Different Planting Regions and Climate Factors Using a Pseudotargeted Method Based on Gas Chromatography/Mass Spectrometry

An improved pseudotargeted 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 pseudotargeted 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.

Study of polar metabolites in tobacco from different geographical origins by using capillary electrophoresis-mass spectrometry

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.

Analysis of free amino acids in flue-cured tobacco leaves using ultra-high performance liquid chromatography with single quadrupole mass spectrometry.

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 LC with single quadrupole MS and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate precolumn derivatization was developed for the analysis of free amino acids in flue-cured tobacco leaves. Unlike 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 LODs for derivatized amino acids were 0.2-9.7 fmol/μL. Good repeatability with an RSD of 2.5-8.6% and satisfactory intra- and interday 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.

Metabolic profiling of transgenic rice progeny using gas chromatography-mass spectrometry: the effects of gene insertion, tissue culture and breeding

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 transformation processes.

Chip-based nanoflow high performance liquid chromatography coupled to mass spectrometry for profiling of soybean flavonoids

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 (R2, 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.

Metabolomics study of cured tobacco using liquid chromatography with mass spectrometry: method development and its application in investigating the chemical differences of tobacco from three growing regions.

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 semipolar 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, intraday and interday 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. Forty three 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.

Study of metabolite differences of flue-cured tobacco from different regions using a pseudotargeted gas chromatography with mass spectrometry selected-ion monitoring method

A pseudotargeted method based on gas chromatography and mass spectrometry with selected-ion monitoring 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.