通过搭建管式燃烧炉-四极杆质谱仪实验平台检测食品中蛋白质含量,称取各1 g 5种蛋白质含量不同的食品样品,在高温富氧环境下利用管式燃烧炉对食品样品进行充分爆燃,气体产物进入电子轰击(EI)离子源离子化后,直接引入四极杆质量分析器进...通过搭建管式燃烧炉-四极杆质谱仪实验平台检测食品中蛋白质含量,称取各1 g 5种蛋白质含量不同的食品样品,在高温富氧环境下利用管式燃烧炉对食品样品进行充分爆燃,气体产物进入电子轰击(EI)离子源离子化后,直接引入四极杆质量分析器进行检测。利用四极杆质量分析器的选择离子扫描功能,通过扫描NO2+离子峰强度来定量分析氮元素含量,并绘制标准曲线,线性相关系数(R2)为0.999 92,相对标准偏差(RSD)为2.1%~6.1%。利用氮元素含量结合氮-蛋白质转换系数6.25计算得到蛋白质含量。本研究为食品中蛋白质含量的定量分析提供了一种绿色、快速、准确、低成本的检测方法。展开更多
Food allergy is a growing health issue worldwide and the demand for sensitive,robust and high-throughput analytical methods is rising.In recent years,mass spectrometry-based methods have been established for multiple ...Food allergy is a growing health issue worldwide and the demand for sensitive,robust and high-throughput analytical methods is rising.In recent years,mass spectrometry-based methods have been established for multiple food allergen detection.In the present study,a novel method was developed for the simultaneous detection of almond,cashew,peanut,and walnut allergens in bakery foods using liquid chromatography–mass spectrometry.Proteins unique to these four ingredients were extracted,followed by trypsin digestion,quadrupole time-of-flight(Q-TOF)mass spectrometry and bioinformatics analysis.The raw data were processed by de-novo sequencing module plus PEAKS DB(database search)module of the PEAKS software to screen peptides specific to each nut species.The thermal stability and uniqueness of these candidate peptides were further verified using triple quadrupole mass spectrometry(QQQ-MS)in multiple reaction monitoring(MRM)mode.Each nut species was represented by four peptides,all of which were validated for label-free quantification(LFQ).Calibration curves were constructed with good linearity and correlation coefficient(r 2)greater than 0.99.The limits of detection(LODs)were determined to range from 0.11 to 0.31 mg/kg,and were compared with the reference doses proposed by Voluntary Incidental Trace Allergen Labelling(VITAL).The recoveries of the developed method in incurred bakery food matrices ranged from 72.5%to 92.1%with relative standard deviations(RSD)of<5.2%.The detection of undeclared allergens in commercial bakery food samples confirmed the presence of these allergens.In conclusion,this method provides insight into the qualitative and quantitative detection of trace levels of nut allergens in bakery foods.展开更多
建立液相色谱-串联质谱技术测定牛排产品中大豆蛋白含量的方法。首先利用高分辨质谱结合Unitprot数据库,对大豆特异性多肽进行鉴别及筛选,获得可用于大豆准确定性的多肽序列信息,并利用skyline软件将多肽序列转换为离子对信息;其次利用...建立液相色谱-串联质谱技术测定牛排产品中大豆蛋白含量的方法。首先利用高分辨质谱结合Unitprot数据库,对大豆特异性多肽进行鉴别及筛选,获得可用于大豆准确定性的多肽序列信息,并利用skyline软件将多肽序列转换为离子对信息;其次利用液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)对大豆蛋白特异性多肽进行确证,构建大豆蛋白LC-MS/MS定性判别方法;进一步开展用于定量多肽的筛选研究,最终筛选到线性和回收率较好的定量多肽共3条,分别为GSDLVNVR、VSDDEFNNYK、LVFCPQQAEDDK,线性相关系数均达到0.99以上,加标回收率为81.7%~115.8%,精密度小于13%,检出限为0.15%,定量限为0.5%;同时对市售28个牛排进行大豆蛋白含量的测定,构建LC-MS/MS方法测定牛排中大豆蛋白含量的方法。结果表明,该方法特异性好,准确度高,同时可推广应用至其他肉制品。展开更多
The scientific community has shown great interest in the field of mass spectrometry-based proteomics and peptidomics for its applications in biology. Proteomics technologies have evolved to produce large data sets of ...The scientific community has shown great interest in the field of mass spectrometry-based proteomics and peptidomics for its applications in biology. Proteomics technologies have evolved to produce large data sets of proteins or peptides involved in various biologic and disease progression processes generating testable hypothesis for complex biologic questions. This review provides an introduction to relevant topics in proteomics and peptidomics including biologic material selection, sample preparation, separation techniques, peptide fragmentation, post-translational modifications, quantification, bioinformatics, and biomarker discovery and validation. In addition, current literature, remaining challenges, and emerging technologies for proteomics and peptidomics are presented.展开更多
High-sensitivity mass spectrometry approaches using selected reaction monitoring(SRM)or multiple reaction monitoring(MRM)methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics ...High-sensitivity mass spectrometry approaches using selected reaction monitoring(SRM)or multiple reaction monitoring(MRM)methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics in specific biological systems.Both high-sensitivity detection of lowabundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal standards.Currently,there are various ways for preparing standards,including chemical peptide synthesis,cellular protein expression,and cell-free protein or peptide synthesis.Cell-free protein synthesis(CFPS)or in vitro translation(IVT)systems in particular provide high-throughput and low-cost preparation methods,and various cell types and reconstituted forms are now commercially available.Herein,we review the use of such systems for precise and reliable protein quantification.展开更多
背景:非创伤性股骨头坏死是一类以髋关节疼痛、功能障碍进行性加重为主要症状的疾病,严重危害人体健康。它的发病机制、早期诊断和治疗仍是临床骨科医生所面临的重大挑战。目的:采用同位素标记相对和绝对定量(isobaric tags for relativ...背景:非创伤性股骨头坏死是一类以髋关节疼痛、功能障碍进行性加重为主要症状的疾病,严重危害人体健康。它的发病机制、早期诊断和治疗仍是临床骨科医生所面临的重大挑战。目的:采用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)技术,用蛋白质组学筛选可能与非创伤性股骨头坏死相关的关键蛋白。通过分析这些蛋白的表达特性及潜在的生物学功能,为进一步研究非创伤性股骨头坏死病因及分子机制奠定基础。方法:提取非创伤性股骨头坏死患者和健康人血清蛋白,应用iTRAQ技术标记蛋白,联合液相色谱和串联质谱分离和分析肽段。采用Proteome Discoverer 2.1软件对两组人群蛋白进行鉴定和定量分析,获得差异蛋白,再对这些表达差异蛋白进行GO注释、KEGG通路注释和功能富集聚类分析。结果与结论:①质谱鉴定共得到1006个蛋白,通过分析得到137个差异表达蛋白,其中54个蛋白表达上调,83个蛋白表达下调;②与非创伤性股骨头坏死发生相关的表达差异显著的蛋白质包括载脂蛋白B(ApoB)、载脂蛋白A2(ApoA2)、载脂蛋白E(ApoE)、补体C4和C7等;③KEGG信号通路富集分析结果显示,关键靶点的主要信号通路为p53信号通路、转录生长因子β信号通路、扩张型心肌病信号通路;④该结果提示,载脂蛋白与非创伤性股骨头坏死发生密切相关,并发现了与非创伤性股骨头坏死相关的活跃信号通路,为非创伤性股骨头坏死发生及分子机制奠定一定的实验基础。展开更多
Recent studies have shown that CTP may act as a ligand to regulate the activity of its target proteins in many biological processes.However,proteome-wide identification of CTP-binding proteins remains challenging.Here...Recent studies have shown that CTP may act as a ligand to regulate the activity of its target proteins in many biological processes.However,proteome-wide identification of CTP-binding proteins remains challenging.Here,we employed a biotinylated CTP affinity probe coupled with stable isotope labeling by amino acids in cell culture(SILAC)-based quantitative proteomics approach to capture,identify and quantify CTP-binding proteins in human cells.By performing two types of competitive SILAC experiments with high vs.low concentrations of CTP probe(100 vs.10µmol/L)or with CTP probe in the presence of free CTP,we identified 90 potential CTP-binding proteins which are involved in a variety of biological processes,including protein folding,nucleotide binding and cell-cell adhesion.Together,we developed a chemical proteomic method for uncovering the CTP-binding proteins in human cells,which could be widely applicable for profiling CTP-binding proteins in other biological samples.展开更多
文摘通过搭建管式燃烧炉-四极杆质谱仪实验平台检测食品中蛋白质含量,称取各1 g 5种蛋白质含量不同的食品样品,在高温富氧环境下利用管式燃烧炉对食品样品进行充分爆燃,气体产物进入电子轰击(EI)离子源离子化后,直接引入四极杆质量分析器进行检测。利用四极杆质量分析器的选择离子扫描功能,通过扫描NO2+离子峰强度来定量分析氮元素含量,并绘制标准曲线,线性相关系数(R2)为0.999 92,相对标准偏差(RSD)为2.1%~6.1%。利用氮元素含量结合氮-蛋白质转换系数6.25计算得到蛋白质含量。本研究为食品中蛋白质含量的定量分析提供了一种绿色、快速、准确、低成本的检测方法。
基金the Key Science and Technology Program of the Market Supervision Administration of Jiangsu Province(No.KJ196035)the Key Science and Technology Program of the Market Supervision Administration of Nanjing(No.KJ2019043),China.
文摘Food allergy is a growing health issue worldwide and the demand for sensitive,robust and high-throughput analytical methods is rising.In recent years,mass spectrometry-based methods have been established for multiple food allergen detection.In the present study,a novel method was developed for the simultaneous detection of almond,cashew,peanut,and walnut allergens in bakery foods using liquid chromatography–mass spectrometry.Proteins unique to these four ingredients were extracted,followed by trypsin digestion,quadrupole time-of-flight(Q-TOF)mass spectrometry and bioinformatics analysis.The raw data were processed by de-novo sequencing module plus PEAKS DB(database search)module of the PEAKS software to screen peptides specific to each nut species.The thermal stability and uniqueness of these candidate peptides were further verified using triple quadrupole mass spectrometry(QQQ-MS)in multiple reaction monitoring(MRM)mode.Each nut species was represented by four peptides,all of which were validated for label-free quantification(LFQ).Calibration curves were constructed with good linearity and correlation coefficient(r 2)greater than 0.99.The limits of detection(LODs)were determined to range from 0.11 to 0.31 mg/kg,and were compared with the reference doses proposed by Voluntary Incidental Trace Allergen Labelling(VITAL).The recoveries of the developed method in incurred bakery food matrices ranged from 72.5%to 92.1%with relative standard deviations(RSD)of<5.2%.The detection of undeclared allergens in commercial bakery food samples confirmed the presence of these allergens.In conclusion,this method provides insight into the qualitative and quantitative detection of trace levels of nut allergens in bakery foods.
文摘建立液相色谱-串联质谱技术测定牛排产品中大豆蛋白含量的方法。首先利用高分辨质谱结合Unitprot数据库,对大豆特异性多肽进行鉴别及筛选,获得可用于大豆准确定性的多肽序列信息,并利用skyline软件将多肽序列转换为离子对信息;其次利用液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)对大豆蛋白特异性多肽进行确证,构建大豆蛋白LC-MS/MS定性判别方法;进一步开展用于定量多肽的筛选研究,最终筛选到线性和回收率较好的定量多肽共3条,分别为GSDLVNVR、VSDDEFNNYK、LVFCPQQAEDDK,线性相关系数均达到0.99以上,加标回收率为81.7%~115.8%,精密度小于13%,检出限为0.15%,定量限为0.5%;同时对市售28个牛排进行大豆蛋白含量的测定,构建LC-MS/MS方法测定牛排中大豆蛋白含量的方法。结果表明,该方法特异性好,准确度高,同时可推广应用至其他肉制品。
文摘The scientific community has shown great interest in the field of mass spectrometry-based proteomics and peptidomics for its applications in biology. Proteomics technologies have evolved to produce large data sets of proteins or peptides involved in various biologic and disease progression processes generating testable hypothesis for complex biologic questions. This review provides an introduction to relevant topics in proteomics and peptidomics including biologic material selection, sample preparation, separation techniques, peptide fragmentation, post-translational modifications, quantification, bioinformatics, and biomarker discovery and validation. In addition, current literature, remaining challenges, and emerging technologies for proteomics and peptidomics are presented.
基金This work was supported by a Grant-in-Aid in number 17H05680(YS)from Japan Society for the Promotion of Science(JSPS)the strategic programs for R&D(President's discretionary fund)of RIKEN(YS)an intramural Grant-in-Aid from the RIKEN Quantitative Biology Center(YS).
文摘High-sensitivity mass spectrometry approaches using selected reaction monitoring(SRM)or multiple reaction monitoring(MRM)methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics in specific biological systems.Both high-sensitivity detection of lowabundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal standards.Currently,there are various ways for preparing standards,including chemical peptide synthesis,cellular protein expression,and cell-free protein or peptide synthesis.Cell-free protein synthesis(CFPS)or in vitro translation(IVT)systems in particular provide high-throughput and low-cost preparation methods,and various cell types and reconstituted forms are now commercially available.Herein,we review the use of such systems for precise and reliable protein quantification.
文摘背景:非创伤性股骨头坏死是一类以髋关节疼痛、功能障碍进行性加重为主要症状的疾病,严重危害人体健康。它的发病机制、早期诊断和治疗仍是临床骨科医生所面临的重大挑战。目的:采用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)技术,用蛋白质组学筛选可能与非创伤性股骨头坏死相关的关键蛋白。通过分析这些蛋白的表达特性及潜在的生物学功能,为进一步研究非创伤性股骨头坏死病因及分子机制奠定基础。方法:提取非创伤性股骨头坏死患者和健康人血清蛋白,应用iTRAQ技术标记蛋白,联合液相色谱和串联质谱分离和分析肽段。采用Proteome Discoverer 2.1软件对两组人群蛋白进行鉴定和定量分析,获得差异蛋白,再对这些表达差异蛋白进行GO注释、KEGG通路注释和功能富集聚类分析。结果与结论:①质谱鉴定共得到1006个蛋白,通过分析得到137个差异表达蛋白,其中54个蛋白表达上调,83个蛋白表达下调;②与非创伤性股骨头坏死发生相关的表达差异显著的蛋白质包括载脂蛋白B(ApoB)、载脂蛋白A2(ApoA2)、载脂蛋白E(ApoE)、补体C4和C7等;③KEGG信号通路富集分析结果显示,关键靶点的主要信号通路为p53信号通路、转录生长因子β信号通路、扩张型心肌病信号通路;④该结果提示,载脂蛋白与非创伤性股骨头坏死发生密切相关,并发现了与非创伤性股骨头坏死相关的活跃信号通路,为非创伤性股骨头坏死发生及分子机制奠定一定的实验基础。
基金supported by the National Natural Science Foundation of China(Nos.21807030,21907028)the Science and Technology Innovation Program of Hunan Province(No.2019RS2020)+1 种基金Natural Science Foundation of Hunan Province(No.2020JJ5046)the Fundamental Research Funds for the Central Universities(Nos.531118010061,531118010259).
文摘Recent studies have shown that CTP may act as a ligand to regulate the activity of its target proteins in many biological processes.However,proteome-wide identification of CTP-binding proteins remains challenging.Here,we employed a biotinylated CTP affinity probe coupled with stable isotope labeling by amino acids in cell culture(SILAC)-based quantitative proteomics approach to capture,identify and quantify CTP-binding proteins in human cells.By performing two types of competitive SILAC experiments with high vs.low concentrations of CTP probe(100 vs.10µmol/L)or with CTP probe in the presence of free CTP,we identified 90 potential CTP-binding proteins which are involved in a variety of biological processes,including protein folding,nucleotide binding and cell-cell adhesion.Together,we developed a chemical proteomic method for uncovering the CTP-binding proteins in human cells,which could be widely applicable for profiling CTP-binding proteins in other biological samples.
