目的建立一种气相色谱-负化学离子源质谱法(gas chromatography-negative chemical ionization-mass spectrometry,GC-NCI/MS)测定白菜中6种痕量农药残留的分析方法。方法采用乙腈超声提取,Carb/氨丙基固相萃取柱净化,酮麝香为内标物,...目的建立一种气相色谱-负化学离子源质谱法(gas chromatography-negative chemical ionization-mass spectrometry,GC-NCI/MS)测定白菜中6种痕量农药残留的分析方法。方法采用乙腈超声提取,Carb/氨丙基固相萃取柱净化,酮麝香为内标物,进行气相色谱–负化学离子源质谱分析检测。结果6种农药残留在相应线性浓度范围内线性良好,回归系数r^2≥0.999,不同浓度区间加标平均回收率在89.2%~111.5%,相对标准偏差(relative standard deviation,RSD)为0.6%~12.7%。五氯硝基苯、百菌清、甲基对硫磷、α-硫丹、β-硫丹、硫丹硫酸酯6种农药残留检出下限分别为0.013、0.06、0.06、0.024、0.012、0.01 ng/mL。结论该方法回收率高,分离效果好,灵敏度高,检出下限低,快速方便,适用于实际蔬菜样品中多农药残留的痕量分析。展开更多
Background Site A132Arg mutations potentially impair the affinity of isocitrate dehydrogenase 1 （IDH1） for its substrate isocitrate （ICT）, consequently reducing the production of a-ketoglutarate and leading to tum...Background Site A132Arg mutations potentially impair the affinity of isocitrate dehydrogenase 1 （IDH1） for its substrate isocitrate （ICT）, consequently reducing the production of a-ketoglutarate and leading to tumor growth through the induction of the hypoxia-inducible factor-1 （HIF-1） pathway. However, given that the roles of other active sites in IDH1 substrate binding remain unclear, we aimed to investigate IDH1 mutation pattern and its influence on enzyme function. Methods Fifteen IDH1 catalytic active site candidates were selected for in silico mutagenesis and protein homology modeling. Binding free energy of the IDH1/ICT complexes with single-site mutations was compared with that of the wild type. The affinity of 10 IDH1 catalytic active sites for the ICT substrate was further calculated. Results The IDH1 active site included seven residues from chain A （A77Thr, A94Ser, A100Arg, A132Arg, A109Arg, A275Asp, and A279Asp） and three residues from chain B （B214Thr, B212Lys, and B252Asp） that constituted the substrate ICT-binding site. These residues were located within 0.5 nm of ICT, indicating a potential interaction with the substrate. IDH1 changes of binding free energy （AE） suggested that the A132Arg residue from chain A contributes three hydrogen bonds to the ICT a-carboxyl and 13-carboxyl groups, while the other nine residues involved in ICT binding form only one or two hydrogen bonds. Amino acid substitutes at A132Arg, A109Arg, and B212Lys sites, had the greatest effect on enzyme affinity for its substrate. Conclusions Mutations at sites A132Arg, A109Arg, and B212Lys reduced IDH1 affinity for ICT, indicating these active sites may play a central role in substrate binding. Mutations at sites A77Thr, A94Ser, and A275Asp increased the affinity of IDH1 for ICT, which may enhance IDN1 catalytic activity. Mutant IDH1 proteins with higher catalytic activity than the wild-tvDe IDH1 could potentially be used as a novel qene therapy for qlioblastoma multiforme.展开更多
文摘目的建立一种气相色谱-负化学离子源质谱法(gas chromatography-negative chemical ionization-mass spectrometry,GC-NCI/MS)测定白菜中6种痕量农药残留的分析方法。方法采用乙腈超声提取,Carb/氨丙基固相萃取柱净化,酮麝香为内标物,进行气相色谱–负化学离子源质谱分析检测。结果6种农药残留在相应线性浓度范围内线性良好,回归系数r^2≥0.999,不同浓度区间加标平均回收率在89.2%~111.5%,相对标准偏差(relative standard deviation,RSD)为0.6%~12.7%。五氯硝基苯、百菌清、甲基对硫磷、α-硫丹、β-硫丹、硫丹硫酸酯6种农药残留检出下限分别为0.013、0.06、0.06、0.024、0.012、0.01 ng/mL。结论该方法回收率高,分离效果好,灵敏度高,检出下限低,快速方便,适用于实际蔬菜样品中多农药残留的痕量分析。
文摘Background Site A132Arg mutations potentially impair the affinity of isocitrate dehydrogenase 1 （IDH1） for its substrate isocitrate （ICT）, consequently reducing the production of a-ketoglutarate and leading to tumor growth through the induction of the hypoxia-inducible factor-1 （HIF-1） pathway. However, given that the roles of other active sites in IDH1 substrate binding remain unclear, we aimed to investigate IDH1 mutation pattern and its influence on enzyme function. Methods Fifteen IDH1 catalytic active site candidates were selected for in silico mutagenesis and protein homology modeling. Binding free energy of the IDH1/ICT complexes with single-site mutations was compared with that of the wild type. The affinity of 10 IDH1 catalytic active sites for the ICT substrate was further calculated. Results The IDH1 active site included seven residues from chain A （A77Thr, A94Ser, A100Arg, A132Arg, A109Arg, A275Asp, and A279Asp） and three residues from chain B （B214Thr, B212Lys, and B252Asp） that constituted the substrate ICT-binding site. These residues were located within 0.5 nm of ICT, indicating a potential interaction with the substrate. IDH1 changes of binding free energy （AE） suggested that the A132Arg residue from chain A contributes three hydrogen bonds to the ICT a-carboxyl and 13-carboxyl groups, while the other nine residues involved in ICT binding form only one or two hydrogen bonds. Amino acid substitutes at A132Arg, A109Arg, and B212Lys sites, had the greatest effect on enzyme affinity for its substrate. Conclusions Mutations at sites A132Arg, A109Arg, and B212Lys reduced IDH1 affinity for ICT, indicating these active sites may play a central role in substrate binding. Mutations at sites A77Thr, A94Ser, and A275Asp increased the affinity of IDH1 for ICT, which may enhance IDN1 catalytic activity. Mutant IDH1 proteins with higher catalytic activity than the wild-tvDe IDH1 could potentially be used as a novel qene therapy for qlioblastoma multiforme.
