Chromatographic fingerprinting has been perceived as an essential tool for assessing quality and chemical equivalence of traditional Chinese medicine.However,this pattern-oriented approach still has some weak points i...Chromatographic fingerprinting has been perceived as an essential tool for assessing quality and chemical equivalence of traditional Chinese medicine.However,this pattern-oriented approach still has some weak points in terms of chemical coverage and robustness.In this work,we proposed a multiple reaction monitoring(MRM)-based fingerprinting method in which approximately 100 constituents were simultaneously detected for quality assessment.The derivative MRM approach was employed to rapidly design MRM transitions independent of chemical standards,based on which the large-scale fingerprinting method was efficiently established.This approach was exemplified on QiShenYiQi Pill(QSYQ),a traditional Chinese medicine-derived drug product,and its robustness was systematically evaluated by four indices:clustering analysis by principal component analysis,similarity analysis by the congruence coefficient,the number of separated peaks,and the peak area proportion of separated peaks.Compared with conventional ultraviolet-based fingerprints,the MRM fingerprints provided not only better discriminatory capacity for the tested normal/abnormal QSYQ samples,but also higher robustness under different chromatographic conditions(i.e.,flow rate,apparent pH,column temperature,and column).The result also showed for such large-scale fingerprints including a large number of peaks,the angle cosine measure after min-max normalization was more suitable for setting a decision criterion than the unnormalized algorithm.This proof-of-concept application gives evidence that combining MRM technique with proper similarity analysis metrices can provide a highly sensitive,robust and comprehensive analytical approach for quality assessment of traditional Chinese medicine.展开更多
Synaptic dysfunction occurs early in Alzheimer＇s disease （AD） and is acknowledged as a primary pathologic target for treatment. Synaptic degeneration is the pathological feature most strongly correlated with loss o...Synaptic dysfunction occurs early in Alzheimer＇s disease （AD） and is acknowledged as a primary pathologic target for treatment. Synaptic degeneration is the pathological feature most strongly correlated with loss of cognitive function ante mortern （Terry et al., 1991）. Synapses are heavily damaged in hippocampal and neocortical regions of AD brain, whereas motor and occipital cortices are relatively spared （Honer et al., 1992）. Despite extensive work, the molecular mechanisms underlying synaptic degeneration are largely unknown.展开更多
Attributing to the rapid demand expansion for the edible medicinal materials in the market,the limited throughput of highperformance liquid chromatography-multiple reaction monitoring(HPLC-MRM)cannot fully address the...Attributing to the rapid demand expansion for the edible medicinal materials in the market,the limited throughput of highperformance liquid chromatography-multiple reaction monitoring(HPLC-MRM)cannot fully address the measurement workload for a huge number of testing samples.Hence,it is urgent to pursue more efficient approaches for the quality evaluation.Because of the greater selectivity of MRM cubed(MRM^(3))over MRM,there might be a chance to omit the time-intensive LC separation.In current study,we attempted to develop a direct infusion(DI)-MRM^(3) program,and the applicability was thereafter assessed through simultaneous determination of four ganoderic acids(GAs)in one of the most famous tonic herbal medicines namely Ganoderma(Chinese name:Lingzhi).Primary parameters such as Q1>Q3>QLIT ion transitions,collision energy(CE),and excitation energy were optimized by programming online energy-resolved mass spectrometry with authentic compounds.A single DI-MRM measurement merely costed four minutes,and in spite of the wide occurrences of isomers,satisfactory selectivity was achieved.Method validation assays demonstrated the method to be sensitive,precise,accurate,and reproducible.The quantitative results from DI-MRM^(3) were also justified by conducting LC-MRM measurements in parallel.Significant differences occurred for the content patterns between the two original sources namely Ganoderma lucidum and G.sinense,and,moreover,either cultivar or harvest time showed dramatical influence on the quantitative features of the four targeted GAs.More importantly,DI-MRM3 is a meaningful analytical option for rapid quantitative analysis of herbal medicines,because of the comparable reliability,nonetheless,less consumptions of both measurement time and solvent,compared with LC-MRM.展开更多
基金financially supported by the National Natural Science Foundation of China(Grant No.81803714)the Fundamental Research Funds for the Central Universities(Grant No.2019QNA7041).
文摘Chromatographic fingerprinting has been perceived as an essential tool for assessing quality and chemical equivalence of traditional Chinese medicine.However,this pattern-oriented approach still has some weak points in terms of chemical coverage and robustness.In this work,we proposed a multiple reaction monitoring(MRM)-based fingerprinting method in which approximately 100 constituents were simultaneously detected for quality assessment.The derivative MRM approach was employed to rapidly design MRM transitions independent of chemical standards,based on which the large-scale fingerprinting method was efficiently established.This approach was exemplified on QiShenYiQi Pill(QSYQ),a traditional Chinese medicine-derived drug product,and its robustness was systematically evaluated by four indices:clustering analysis by principal component analysis,similarity analysis by the congruence coefficient,the number of separated peaks,and the peak area proportion of separated peaks.Compared with conventional ultraviolet-based fingerprints,the MRM fingerprints provided not only better discriminatory capacity for the tested normal/abnormal QSYQ samples,but also higher robustness under different chromatographic conditions(i.e.,flow rate,apparent pH,column temperature,and column).The result also showed for such large-scale fingerprints including a large number of peaks,the angle cosine measure after min-max normalization was more suitable for setting a decision criterion than the unnormalized algorithm.This proof-of-concept application gives evidence that combining MRM technique with proper similarity analysis metrices can provide a highly sensitive,robust and comprehensive analytical approach for quality assessment of traditional Chinese medicine.
基金Financial support was provided by the Alzheimer’s Australia Dementia Research Foundation Scholarship Program(AAR Postgraduate Research Scholarship),Alzheimer’s Association(USA)under grant#RG1-96-005the Judith Jane Mason and Harold Stannett Williams Memorial Foundation+1 种基金The Queensland Brain Bank,part of Australian Brain Bank Networksupported by an NHMRC(Australia)Enabling Grant No.605210
文摘Synaptic dysfunction occurs early in Alzheimer＇s disease （AD） and is acknowledged as a primary pathologic target for treatment. Synaptic degeneration is the pathological feature most strongly correlated with loss of cognitive function ante mortern （Terry et al., 1991）. Synapses are heavily damaged in hippocampal and neocortical regions of AD brain, whereas motor and occipital cortices are relatively spared （Honer et al., 1992）. Despite extensive work, the molecular mechanisms underlying synaptic degeneration are largely unknown.
基金financially supported by the National Natural Science Foundation of China(81973444)the Open Research Project Programme of the State Key Laboratory of Quality Research in Chinese Medicine(University of Macao)(SKLQRCM-OP21011).
文摘Attributing to the rapid demand expansion for the edible medicinal materials in the market,the limited throughput of highperformance liquid chromatography-multiple reaction monitoring(HPLC-MRM)cannot fully address the measurement workload for a huge number of testing samples.Hence,it is urgent to pursue more efficient approaches for the quality evaluation.Because of the greater selectivity of MRM cubed(MRM^(3))over MRM,there might be a chance to omit the time-intensive LC separation.In current study,we attempted to develop a direct infusion(DI)-MRM^(3) program,and the applicability was thereafter assessed through simultaneous determination of four ganoderic acids(GAs)in one of the most famous tonic herbal medicines namely Ganoderma(Chinese name:Lingzhi).Primary parameters such as Q1>Q3>QLIT ion transitions,collision energy(CE),and excitation energy were optimized by programming online energy-resolved mass spectrometry with authentic compounds.A single DI-MRM measurement merely costed four minutes,and in spite of the wide occurrences of isomers,satisfactory selectivity was achieved.Method validation assays demonstrated the method to be sensitive,precise,accurate,and reproducible.The quantitative results from DI-MRM^(3) were also justified by conducting LC-MRM measurements in parallel.Significant differences occurred for the content patterns between the two original sources namely Ganoderma lucidum and G.sinense,and,moreover,either cultivar or harvest time showed dramatical influence on the quantitative features of the four targeted GAs.More importantly,DI-MRM3 is a meaningful analytical option for rapid quantitative analysis of herbal medicines,because of the comparable reliability,nonetheless,less consumptions of both measurement time and solvent,compared with LC-MRM.
文摘目的构建膀胱尿路上皮癌(bladder urothelial carcinoma,BUC)尿液差异蛋白数据集,筛选核心差异蛋白并初步验证。方法于2015年1月至2016年11月在天津医科大学第二医院泌尿外科住院病房,采集膀胱上皮癌患者术前尿液标本78例,其中男性55人,女性23人,年龄(68.9±6.6)岁。同时收集51例健康志愿者尿液标本用于对照组分析,其中男性34人,女性17人,年龄(60.6±11.0)岁。采用同位素相对标记和绝对定量技术(isobaric tags for relative and absolute quantitation,iTRAQ)对BUC组混合尿液样本4例和对照组混合尿液样本2例,共6例尿液混合标本进行比较蛋白质组学研究,找出两组间的尿液差异蛋白,选取差异倍数前20的差异蛋白进行质谱多反应监测(multiple reaction monitoring,MRM)技术验证;应用R语言cluster profiler包等进行差异蛋白的京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)富集分析、应用R语言的org.Hs.eg.db包等进行基因本体(Gene ontology,GO)分析,初步选取有意义的差异蛋白,构建BUC尿液差异蛋白数据集。应用R语言绘制蛋白间相互作用(protein protein interaction,PPI)图,筛选核心差异蛋白。用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)验证核心差异蛋白在尿液中的差异存在。结果通过分析得到101个BUC尿液差异蛋白,其中37个蛋白表达上调,64个蛋白表达下调。MRM检测到10个差异蛋白;KEGG分析显示差异蛋白显著富集在其他聚糖降解,补体和凝血级联这两个代谢通路,包含11个差异蛋白;GO分析显示差异蛋白主要参与细胞黏附分子结合、羧酸结合、有机酸结合、糖胺聚糖结合、肽链内切酶激活等生物过程,涉及54个差异蛋白;三者共同构成BUC尿液差异蛋白数据集,包括69个蛋白。通过蛋白互作分析筛选出APOE和APOA4为核心差异蛋白。ELISA结果显示:BUC组和对照组的APOE平均浓度分别为0.55和0.30 pg/mL。BUC组和对照组的APOA4平均浓度分别为3.33和7.01 ng/mL。与对照组相比,BUC组尿液中APOE和APOA4浓度均增高,差异有统计显著性(P<0.05)。结论应用iTRAQ-MRM-R语言策略可以辅助构建BUC尿液差异蛋白数据集,并进一步筛选出核心差异蛋白,为BUC的诊断及治疗提供新的靶点。
