Monitoring of host cell proteins(HCPs)during the manufacturing of monoclonal antibodies(mAb)has become a critical requirement to provide effective and safe drug products.Enzyme-linked immunosorbent assays are still th...Monitoring of host cell proteins(HCPs)during the manufacturing of monoclonal antibodies(mAb)has become a critical requirement to provide effective and safe drug products.Enzyme-linked immunosorbent assays are still the gold standard methods for the quantification of protein impurities.However,this technique has several limitations and does,among others,not enable the precise identification of proteins.In this context,mass spectrometry(MS)became an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs.However,in order to be routinely implemented in biopharmaceutical companies,liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification.Here,we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard,the HCP Profiler solution,with a spectral library-based data-independent acquisition(DIA)method and strict data validation criteria.The performances of the HCP Profiler solution were compared to more conventional standard protein spikes and the DIA approach was benchmarked against a classical datadependent acquisition on a series of samples produced at various stages of the manufacturing process.While we also explored spectral library-free DIA interpretation,the spectral library-based approach still showed highest accuracy and reproducibility(coefficients of variation<10%)with a sensitivity down to the sub-ng/mg mAb level.Thus,this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.展开更多
The main advantage of digital PCR(dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve.Crystal droplet dPCR has a three-color staining detection functi...The main advantage of digital PCR(dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve.Crystal droplet dPCR has a three-color staining detection function,which enables multiplex PCR reaction.In this study,this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708 × MON89788 with stacked traits.Specific absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708× MON89788 soybean.Our results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.展开更多
The CP4-EPSPS gene is widely used in herbicide-tolerant plants/crops all over the world. In this study, a method was developed by coupling liquid chromatography with high sensitivity to tandem mass spectrometry to qua...The CP4-EPSPS gene is widely used in herbicide-tolerant plants/crops all over the world. In this study, a method was developed by coupling liquid chromatography with high sensitivity to tandem mass spectrometry to quantify the amount of CP4-EPSPS expression in Nicotiana tabacum leaves. The quantification of protein was converted to measure the unique peptide of CP4-EPSPS protein. One peptide unique to CP4-EPSPS was synthesized and labeled with.H2^18O to get 180 stable isotope labeled peptide. The peptide served as the internal standard. The validated method had good specificity and linearity. The intra-and inter-day precisions and accuracy for all samples were satisfactory. The results demonstrated that the novel method was sensitive and selective to quantify CP4- EPSPS in the crude extract without time-consuming pre-separation or.the purification procedures.展开更多
目的筛选家系早发冠心病血瘀证的特异性蛋白,为今后临床早期诊断和预防疾病提供潜在生物标志物。方法通过同位素相对标记与绝对定量技术(isobaric tags for relative and absolute quantification,i TRAQ)分析家系早发冠心病血瘀证患者...目的筛选家系早发冠心病血瘀证的特异性蛋白,为今后临床早期诊断和预防疾病提供潜在生物标志物。方法通过同位素相对标记与绝对定量技术(isobaric tags for relative and absolute quantification,i TRAQ)分析家系早发冠心病血瘀证患者、家系早发冠心病非血瘀证患者及健康人的血浆蛋白表达,数据分析后得到各组间的差异蛋白表达情况,再经差异蛋白筛选标准初步得到疾病的预测蛋白,并使用Western blot验证预测蛋白。结果通过i TRAQ技术共得到差异蛋白75个,其中家系早发冠心病血瘀证组与健康对照组之间共得到32个差异蛋白,包括22个上调蛋白与10个下调蛋白,共涉及429个生物学过程,其与角质化、皮肤发展、蛋白质激活级联反应等关系最为密切;在代谢途径金黄色葡萄球菌感染、补体和凝血级联、血小板激活等KEGG通路上富集。与健康对照组相比,家系早发冠心病血瘀证组差异蛋白补体因子H(complement factor H,CFH)表达水平差异有统计学意义(P<0.01)。结论经差异蛋白筛选标准得到的CFH蛋白,可作为家系早发冠心病血瘀证早期诊断的潜在生物标志物。展开更多
With worldwide attention on renewable energy and climate change,metabolic engineering of the fatty acid biosynthetic pathway has become an active area of research,with a view to enhance production of biofuels.Indeed,t...With worldwide attention on renewable energy and climate change,metabolic engineering of the fatty acid biosynthetic pathway has become an active area of research,with a view to enhance production of biofuels.Indeed,this pathway has already been extensively studied in Escherichia coli.Nevertheless,little is known about the absolute abundance of the enzymes involved,information that may be valuable for engineering,such as the optimal molar ratios of different proteins.In this study,we use protein standard absolute quantification(PSAQ)to measure the absolute abundance of proteins that catalyze fatty acid biosynthesis in E.coli.In addition,the changes of protein abundance were analyzed by comparing the differences between high-yield and the background strain.Our work highlights opportunities to enhance fatty acid production by measuring protein molar ratios and identifying catalytic and regulatory bottlenecks.More importantly,our results provide evidence that PSAQ is a generally valuable tool to investigate metabolic pathways.展开更多
目的应用同位素标记相对与绝对定量蛋白组学(isobaric tags for relative and absolute quantification,iTRAQ)技术鉴定和筛选特发性膜性肾病(idiopathic membranous nephropathy,IMN)患者外周血中的差异蛋白,验证IMN患者差异蛋白S100A...目的应用同位素标记相对与绝对定量蛋白组学(isobaric tags for relative and absolute quantification,iTRAQ)技术鉴定和筛选特发性膜性肾病(idiopathic membranous nephropathy,IMN)患者外周血中的差异蛋白,验证IMN患者差异蛋白S100A8表达水平及其意义。方法收集IMN组、非膜性肾病组(not-idiopathic membranous nephropathy,NIMN)和健康对照组的外周血标本,结合生物信息学分析获得差异表达蛋白。采用蛋白质免疫印迹(Western Blot,WB)及酶联免疫吸附方法(ELISA法)检测S100A8蛋白在3组的表达,分析其表达与IMN的关系。结果①通过iTRAQ方法并结合生物信息学分析筛选出S100A8作为目标蛋白;②S100A8在30例IMN患者中表达增高,与NIMN组差异有统计学意义(P<0.001);③ROC曲线显示S100A8蛋白对IMN有高诊断价值。结论检测S100A8蛋白的表达对诊断IMN具有一定价值,S100A8可能是IMN的候选生物标志物。展开更多
基金supported by the“Association Nationale de la Recherche et de la Technologie”and UCB Pharma S.A.(Belgium and France)via the CIFRE fellowship of Steve Hessmannsupported by the“Agence Nationale de la Recherche”via the French Proteomic Infrastructure ProFI FR2048(ANR-10-INBS-08-03).
文摘Monitoring of host cell proteins(HCPs)during the manufacturing of monoclonal antibodies(mAb)has become a critical requirement to provide effective and safe drug products.Enzyme-linked immunosorbent assays are still the gold standard methods for the quantification of protein impurities.However,this technique has several limitations and does,among others,not enable the precise identification of proteins.In this context,mass spectrometry(MS)became an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs.However,in order to be routinely implemented in biopharmaceutical companies,liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification.Here,we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard,the HCP Profiler solution,with a spectral library-based data-independent acquisition(DIA)method and strict data validation criteria.The performances of the HCP Profiler solution were compared to more conventional standard protein spikes and the DIA approach was benchmarked against a classical datadependent acquisition on a series of samples produced at various stages of the manufacturing process.While we also explored spectral library-free DIA interpretation,the spectral library-based approach still showed highest accuracy and reproducibility(coefficients of variation<10%)with a sensitivity down to the sub-ng/mg mAb level.Thus,this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.
文摘The main advantage of digital PCR(dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve.Crystal droplet dPCR has a three-color staining detection function,which enables multiplex PCR reaction.In this study,this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708 × MON89788 with stacked traits.Specific absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708× MON89788 soybean.Our results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.
基金Supported by National Natural Science Foundation of China(21205005,81471919,21475010)MOST China(2011YQ0900502)+1 种基金1000 PlanResearch Foundation of China CDC(2014A101)
文摘The CP4-EPSPS gene is widely used in herbicide-tolerant plants/crops all over the world. In this study, a method was developed by coupling liquid chromatography with high sensitivity to tandem mass spectrometry to quantify the amount of CP4-EPSPS expression in Nicotiana tabacum leaves. The quantification of protein was converted to measure the unique peptide of CP4-EPSPS protein. One peptide unique to CP4-EPSPS was synthesized and labeled with.H2^18O to get 180 stable isotope labeled peptide. The peptide served as the internal standard. The validated method had good specificity and linearity. The intra-and inter-day precisions and accuracy for all samples were satisfactory. The results demonstrated that the novel method was sensitive and selective to quantify CP4- EPSPS in the crude extract without time-consuming pre-separation or.the purification procedures.
基金grants from the 863(2012AA02A701)973(2011CBA00800,2012CB721000)Project from the Ministry of Science and Technology of the People’s Republic of China and the National Natural Science Foundation of China(31170096,31222002)as well as the project from Key Laboratory of Biofuels,Qingdao Institute of Bioenergy and Bioprocess Technology,Chinese Academy of Sciences(CASKLB201301)Science and Technology Department of Hubei Province and J1 Biotech Co.Ltd.(2014091610010595).
文摘With worldwide attention on renewable energy and climate change,metabolic engineering of the fatty acid biosynthetic pathway has become an active area of research,with a view to enhance production of biofuels.Indeed,this pathway has already been extensively studied in Escherichia coli.Nevertheless,little is known about the absolute abundance of the enzymes involved,information that may be valuable for engineering,such as the optimal molar ratios of different proteins.In this study,we use protein standard absolute quantification(PSAQ)to measure the absolute abundance of proteins that catalyze fatty acid biosynthesis in E.coli.In addition,the changes of protein abundance were analyzed by comparing the differences between high-yield and the background strain.Our work highlights opportunities to enhance fatty acid production by measuring protein molar ratios and identifying catalytic and regulatory bottlenecks.More importantly,our results provide evidence that PSAQ is a generally valuable tool to investigate metabolic pathways.
