Fatty acids(FAs)are an important class of functional small molecules and participate in a variety of life biological processes.This experiment proposed a liquid chromatography–quadrupole-Qrbitrap mass spectrometry(LC...Fatty acids(FAs)are an important class of functional small molecules and participate in a variety of life biological processes.This experiment proposed a liquid chromatography–quadrupole-Qrbitrap mass spectrometry(LC–Q-Orbitrap MS)method to perform FA profiling under parallel reaction monitoring(PRM)acquisition mode.The FA was derivatized by 2-dimethylaminoethylamine(DMED)to increase the ionization e fficiency and provide the characterized fragment pattern.The mass spectra obtained from full scan,multiple ion monitoring(MIM),and PRM were compared.The results showed that the protonated ion of FA+DMED–H2O was detected and the neutral loss of 45.06 Da was observed in the tandem mass spectrum.The PRM method provided the highest selectivity and sensitivity for FA detection with the help of accurate mass weight and specific neutral loss-based fragments.The method validation was performed using the FA standards and pooled quality control serum sample,which showed that the established method had good repeatability,stability,and linearity.Finally,the developed method was successfully applied to analyze the rat serum and brain tissue samples for the drug e ffi-cacy evaluation of gross saponin of Tribulus terrestris L.fruit(GSTTF)against middle cerebral artery occlusion(MCAO)-induced ischemic stroke.This method has extensive practicability and great potential in the detection of FA,especially for the analysis of samples with complex matrices.展开更多
Ogura cytoplasmic male sterility(Ogura CMS)is extensively applied in hybrid seed production in cruciferous crops.However,the posttranscriptional molecular basis of Ogura CMS in cruciferous crops remains elusive.Here,a...Ogura cytoplasmic male sterility(Ogura CMS)is extensively applied in hybrid seed production in cruciferous crops.However,the posttranscriptional molecular basis of Ogura CMS in cruciferous crops remains elusive.Here,a data-independent acquisition-based proteomic approach coupled with a parallel reaction monitoring-based targeted proteomic assay was used to analyze the proteome dynamics of Ogura CMS cabbage line RM and its maintainer line RF during floral bud development to obtain insights into the mechanism underlying Ogura CMS in cruciferous crops.A total of 9162 proteins corresponding to 61464 peptides were identified in RM and RF floral buds.The proteomic fluctuation of RM was weaker than that of RF.Differences in protein expression between RM and RF gradually enlarged with floral bud development.Fifteen continually up-regulated and eight continually down-regulated proteins were found in RM relative to RF throughout floral bud development.Differentially expressed proteins between RM and RF during floral bud development were implicated in the endoplasmic reticulum(ER)-associated protein processing pathway,in which most of them exhibited down-regulated expression in RM.These data suggest that ER-associated protein processing may be involved in pollen abortion in Ogura CMS cabbage by inhibiting the expression of critical factors.Our findings not only deepen the understanding of the molecular mechanisms of Ogura CMS in cruciferous crops but also provide better guidance for applying Ogura CMS in the hybrid breeding of cruciferous crops.展开更多
【目的】系统研究棉秆的化学成分,为其深入开发利用奠定基础。【方法】采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(ultra-high-performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution ma...【目的】系统研究棉秆的化学成分,为其深入开发利用奠定基础。【方法】采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(ultra-high-performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry,UHPLC-Q-Exactive orbitrap MS),色谱柱为Thermo Scientific Hypersil GOLD^(TM)aQ(100 mm×2.1 mm,1.9μm),流动相为0.1%甲酸水溶液(A)-乙腈(B),流速为0.3 m L·min^(-1),柱温为40℃,进样量为2μL;质谱采用电喷雾离子源,在正、负离子模式下采用一级母离子全扫描和数据依赖性前三强二级子离子扫描并结合平行反应监测(parallel reaction monitoring,PRM)模式采集数据。【结果】在棉秆中共鉴定了102种化合物,包括有机酸类化合物48种、黄酮及其苷类化合物13种、萜类化合物10种、核苷酸类化合物8种、氨基酸类化合物8种、香豆素类化合物3种、生物碱类化合物2种、其他化合物10种。其中,92种为首次在棉秆中发现。【结论】建立的UHPLC-Q-Exactive orbitrap MS结合PRM方法操作简便,灵敏度高,分析速度快,鉴定了棉秆中102种化合物,首次鉴定了92种,为棉秆的深入开发利用奠定基础。展开更多
BACKGROUND Colorectal cancer(CRC)is the second leading cause of cancer-related death,with high morbidity worldwide.There is an urgent need to find reliable diagnostic biomarkers of CRC and explore the underlying molec...BACKGROUND Colorectal cancer(CRC)is the second leading cause of cancer-related death,with high morbidity worldwide.There is an urgent need to find reliable diagnostic biomarkers of CRC and explore the underlying molecular mechanisms.Exosomes are involved in intercellular communication and participate in multiple pathological processes,serving as an important part of the tumor microenvironment.AIM To investigate the proteomic characteristics of CRC tumor-derived exosomes and to identify candidate exosomal protein markers for CRC.METHODS In this study,10 patients over 50 years old who were diagnosed with moderately differentiated adenocarcinoma were recruited.We paired CRC tissues and adjacent normal intestinal tissues(>5 cm)to form the experimental and control groups.Purified exosomes were extracted separately from each tissue sample.Data-independent acquisition mass spectrometry was implemented in 8 matched samples of exosomes to explore the proteomic expression profiles,and differentially expressed proteins(DEPs)were screened by bioinformatics analysis.Promising exosomal proteins were verified using parallel reaction monitoring(PRM)analysis in 10 matched exosome samples.RESULTS A total of 1393 proteins were identified in the CRC tissue group,1304 proteins were identified in the adjacent tissue group,and 283 proteins were significantly differentially expressed between them.Enrichment analysis revealed that DEPs were involved in multiple biological processes related to cytoskeleton construction,cell movement and migration,immune response,tumor growth and telomere metabolism,as well as ECM-receptor interaction,focal adhesion and mTOR signaling pathways.Six differentially expressed exosomal proteins(NHP2,OLFM4,TOP1,SAMP,TAGL and TRIM28)were validated by PRM analysis and evaluated by receiver operating characteristic curve(ROC)analysis.The area under the ROC curve was 0.93,0.96,0.97,0.78,0.75,and 0.88(P<0.05)for NHP2,OLFM4,TOP1,SAMP,TAGL,and TRIM28,respectively,indicating their good ability to distinguish CRC tissues from adjacent intestinal tissues.CONCLUSION In our study,comprehensive proteomic profiles were obtained for CRC tissue exosomes.Six exosomal proteins,NHP2,OLFM4,TOP1,SAMP,TAGL and TRIM28,may be promising diagnostic markers and effective therapeutic targets for CRC,but further experimental investigation is needed.展开更多
Xiaoer-Feire-Kechuan(XFK) is an 11-herb Chinese medicine formula to treat cough and pulmonary inflammation.The complicated composition rendered its chemical analysis and effective-component elucidation.In this study,w...Xiaoer-Feire-Kechuan(XFK) is an 11-herb Chinese medicine formula to treat cough and pulmonary inflammation.The complicated composition rendered its chemical analysis and effective-component elucidation.In this study,we combined quantitative analysis and bioactivity test to reveal the antiinflammatory constituents of XFK.First,UPLC-DAD and UHPLC/Q-Orbitrap-MS methods were established and validated to quantify 35 analytes(covering 9 out of 11 herbs) in different XFK formulations.Parallel reaction monitoring mode built in Q-Orbitrap-MS was used to improve the sensitivity and selectivity.Then,anti-inflammatory activities of the 35 analytes were analyzed using in vitro COX-2 inhibition assay.Finally,major analytes forsythosides H,I,A(8-10),and baicalin(15)(total contents varied from 21.79 to 91.20 mg/dose in different formulations) with significant activities(inhibitory rate ≥ 80%) were proposed as the anti-inflammatory constituents of XFK.The present study provided an effective strategy to discover effective constituents of multi-herb formulas.展开更多
旨在对绵羊胚胎骨骼肌蛋白质组学数据进行分析,为阐明绵羊胚胎骨骼肌生长发育机制奠定基础。前期试验应用串联质谱定量法(tandem mass tag,TMT)对成年中国美利奴绵羊妊娠第85(D85)、105(D105)和135天(D135)的胚胎背最长肌进行蛋白质定量...旨在对绵羊胚胎骨骼肌蛋白质组学数据进行分析,为阐明绵羊胚胎骨骼肌生长发育机制奠定基础。前期试验应用串联质谱定量法(tandem mass tag,TMT)对成年中国美利奴绵羊妊娠第85(D85)、105(D105)和135天(D135)的胚胎背最长肌进行蛋白质定量,并对D85/D105、D105/D135和D85/D1353个比较组进行分析。本试验在此基础上,利用KEGG和平行反应监测(parallel reaction monitoring,PRM)等数据分析方法对3个比较组绵羊胚胎骨骼肌上、下调差异丰度蛋白质进行分析和验证,并对候选蛋白质进行生物信息学分析。本研究对肌纤维成熟分化标志性蛋白肌球蛋白重链(myosin-2 isoform X2,MYH)进行PRM靶向定量验证,结果表明,其变化趋势与绵羊胚胎肌纤维成熟分化趋势相符。本研究对上调差异丰度蛋白和下调差异丰度蛋白的KEGG分析结果表明,D105/D85比较组中上调差异丰度蛋白质显著富集于胰岛素等信号通路,D135/D105和D135/D85比较组中下调差异丰度蛋白质显著富集于DNA复制和蛋白质消化吸收等信号通路。与肌肉发育相关的cAMP依赖性蛋白激酶A催化亚基α(PRKACA)和葡萄糖转运蛋白成员4(GLUT4)蛋白均显著富集于胰岛素信号通路中,生物信息学分析显示,这两个蛋白质的理论分子量分别为121.73和20.64 ku,分别有147、12个带正电荷的氨基酸残基和135、12个带负电荷的氨基酸残基;理论等电点分别为8.81和6.54;亲水性平均系数分别为-0.408和0.811;PRKACA蛋白无N端糖基化位点,有45个磷酸化位点;GLUT4蛋白有1个N端糖基化位点,25个磷酸化位点;PRKACA蛋白三级结构与CAMP-2依赖蛋白激酶a嵌合融合的晶体结构相似度为85%,GLUT4蛋白三级结构与人葡萄糖转运蛋白GLUT1的相似性为78%。本研究表明,PRM定量验证趋势与绵羊胚胎肌纤维成熟分化趋势相符,PRKACA和GLUT4蛋白具有丰富的磷酸化修饰位点,并参与胰岛素信号通路调控,是重要的候选蛋白。展开更多
基金supported by the National Natural Science Foundation of China(82104366)the Department of Science and Technology of Jilin Province(20210101426JC and 20220505047ZP)the Changchun University of Chinese Medicine(202326)。
文摘Fatty acids(FAs)are an important class of functional small molecules and participate in a variety of life biological processes.This experiment proposed a liquid chromatography–quadrupole-Qrbitrap mass spectrometry(LC–Q-Orbitrap MS)method to perform FA profiling under parallel reaction monitoring(PRM)acquisition mode.The FA was derivatized by 2-dimethylaminoethylamine(DMED)to increase the ionization e fficiency and provide the characterized fragment pattern.The mass spectra obtained from full scan,multiple ion monitoring(MIM),and PRM were compared.The results showed that the protonated ion of FA+DMED–H2O was detected and the neutral loss of 45.06 Da was observed in the tandem mass spectrum.The PRM method provided the highest selectivity and sensitivity for FA detection with the help of accurate mass weight and specific neutral loss-based fragments.The method validation was performed using the FA standards and pooled quality control serum sample,which showed that the established method had good repeatability,stability,and linearity.Finally,the developed method was successfully applied to analyze the rat serum and brain tissue samples for the drug e ffi-cacy evaluation of gross saponin of Tribulus terrestris L.fruit(GSTTF)against middle cerebral artery occlusion(MCAO)-induced ischemic stroke.This method has extensive practicability and great potential in the detection of FA,especially for the analysis of samples with complex matrices.
基金supported by Heilongjiang Provincial Natural Science Foundation of China(Grant No.YQ2022C012)China Postdoctoral Science Foundation(Grant No.2022MD713728)+1 种基金Heilongjiang Provincial Postdoctoral Fund(Grant No.LBHZ21046)the Open Project of Key Laboratory of Biology and Genetic Improvement of Horticultural Crops(Northeast Region),Ministry of Agriculture and Rural Affairs,and National Key Research and Development Program of China(Grant No.2023YFD1201501).
文摘Ogura cytoplasmic male sterility(Ogura CMS)is extensively applied in hybrid seed production in cruciferous crops.However,the posttranscriptional molecular basis of Ogura CMS in cruciferous crops remains elusive.Here,a data-independent acquisition-based proteomic approach coupled with a parallel reaction monitoring-based targeted proteomic assay was used to analyze the proteome dynamics of Ogura CMS cabbage line RM and its maintainer line RF during floral bud development to obtain insights into the mechanism underlying Ogura CMS in cruciferous crops.A total of 9162 proteins corresponding to 61464 peptides were identified in RM and RF floral buds.The proteomic fluctuation of RM was weaker than that of RF.Differences in protein expression between RM and RF gradually enlarged with floral bud development.Fifteen continually up-regulated and eight continually down-regulated proteins were found in RM relative to RF throughout floral bud development.Differentially expressed proteins between RM and RF during floral bud development were implicated in the endoplasmic reticulum(ER)-associated protein processing pathway,in which most of them exhibited down-regulated expression in RM.These data suggest that ER-associated protein processing may be involved in pollen abortion in Ogura CMS cabbage by inhibiting the expression of critical factors.Our findings not only deepen the understanding of the molecular mechanisms of Ogura CMS in cruciferous crops but also provide better guidance for applying Ogura CMS in the hybrid breeding of cruciferous crops.
基金Supported by National Key Development Plan for Precision Medicine Research,No.2017YFC0910002。
文摘BACKGROUND Colorectal cancer(CRC)is the second leading cause of cancer-related death,with high morbidity worldwide.There is an urgent need to find reliable diagnostic biomarkers of CRC and explore the underlying molecular mechanisms.Exosomes are involved in intercellular communication and participate in multiple pathological processes,serving as an important part of the tumor microenvironment.AIM To investigate the proteomic characteristics of CRC tumor-derived exosomes and to identify candidate exosomal protein markers for CRC.METHODS In this study,10 patients over 50 years old who were diagnosed with moderately differentiated adenocarcinoma were recruited.We paired CRC tissues and adjacent normal intestinal tissues(>5 cm)to form the experimental and control groups.Purified exosomes were extracted separately from each tissue sample.Data-independent acquisition mass spectrometry was implemented in 8 matched samples of exosomes to explore the proteomic expression profiles,and differentially expressed proteins(DEPs)were screened by bioinformatics analysis.Promising exosomal proteins were verified using parallel reaction monitoring(PRM)analysis in 10 matched exosome samples.RESULTS A total of 1393 proteins were identified in the CRC tissue group,1304 proteins were identified in the adjacent tissue group,and 283 proteins were significantly differentially expressed between them.Enrichment analysis revealed that DEPs were involved in multiple biological processes related to cytoskeleton construction,cell movement and migration,immune response,tumor growth and telomere metabolism,as well as ECM-receptor interaction,focal adhesion and mTOR signaling pathways.Six differentially expressed exosomal proteins(NHP2,OLFM4,TOP1,SAMP,TAGL and TRIM28)were validated by PRM analysis and evaluated by receiver operating characteristic curve(ROC)analysis.The area under the ROC curve was 0.93,0.96,0.97,0.78,0.75,and 0.88(P<0.05)for NHP2,OLFM4,TOP1,SAMP,TAGL,and TRIM28,respectively,indicating their good ability to distinguish CRC tissues from adjacent intestinal tissues.CONCLUSION In our study,comprehensive proteomic profiles were obtained for CRC tissue exosomes.Six exosomal proteins,NHP2,OLFM4,TOP1,SAMP,TAGL and TRIM28,may be promising diagnostic markers and effective therapeutic targets for CRC,but further experimental investigation is needed.
基金supported by the National Key Research and Development Program of China (Grant No.: 2018YFC1707304, 2018YFC1707301)Beijing Natural Science Foundation (Grant No.: JQ18027)National Natural Science Foundation of China (Grant No.: 81725023)。
文摘Xiaoer-Feire-Kechuan(XFK) is an 11-herb Chinese medicine formula to treat cough and pulmonary inflammation.The complicated composition rendered its chemical analysis and effective-component elucidation.In this study,we combined quantitative analysis and bioactivity test to reveal the antiinflammatory constituents of XFK.First,UPLC-DAD and UHPLC/Q-Orbitrap-MS methods were established and validated to quantify 35 analytes(covering 9 out of 11 herbs) in different XFK formulations.Parallel reaction monitoring mode built in Q-Orbitrap-MS was used to improve the sensitivity and selectivity.Then,anti-inflammatory activities of the 35 analytes were analyzed using in vitro COX-2 inhibition assay.Finally,major analytes forsythosides H,I,A(8-10),and baicalin(15)(total contents varied from 21.79 to 91.20 mg/dose in different formulations) with significant activities(inhibitory rate ≥ 80%) were proposed as the anti-inflammatory constituents of XFK.The present study provided an effective strategy to discover effective constituents of multi-herb formulas.
