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Synthetic and Systems Biotechnology 2016年第3期1卷 150-157页

作者： Hui Tao Yuchen Zhang Xiaoying Cao Zixin Deng Tiangang Liu Key Laboratory of Combinatorial Biosynthesis and Drug Discovery Ministry of EducationWuhan University School of Pharmaceutical SciencesWuhan 430071P.R.China Hubei Engineering Laboratory for Synthetic Microbiology Wuhan Institute of BiotechnologyWuhan 430075P.R.China State Key Laboratory of Microbial Metabolism School of Life Sciences and BiotechnologyShanghai Jiao Tong University800 Dongchuan RoadShanghai 200240P.R.China Hubei Provincial Cooperative Innovation Center of Industrial Fermentation Wuhan 430068P.R.China

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 ***,this pathway has already been extensively studied in Escherichia ***,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 *** this study,we use protein standard absolute quantification(PSAQ)to measure the absolute abundance of proteins that catalyze fatty acid biosynthesis in *** addition,the changes of protein abundance were analyzed by comparing the differences between high-yield and the background *** work highlights opportunities to enhance fatty acid production by measuring protein molar ratios and identifying catalytic and regulatory *** importantly,our results provide evidence that PSAQ is a generally valuable tool to investigate metabolic pathways.

关键词： Fatty acid absolute quantification Proteomics MRM PSAQ

Fully Automated Protein absolute quantification Platform for Simultaneous Analysis of Multiple Tumor Markers in Human Plasma

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Fully Automated Protein Absolute Quantification Platform for...

High-throughput absolute quantification of Proteins by an Improved Two Dimensional Reversed-phase Separation and quantification Concatamer（QconCAT）Approach

2018年中国质谱学术大会（CMSC2018）

作者： Huiming Yuan Yuanbo Chen Lihua Zhang Yukui Zhang Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical PhysicsChinese Academy of Science

The proteins in the circulatory system reflect the individual’s physiological and disease states. However, many protein biomarkers used for clinical diagnosis are present at very low concentration in body fluids and the detection is very difficult. In this study, we developed a fully automated protein abosolute quantification platform for simultaneous analysis of multiple tumor markers in human plasma, by which alpha-fetoprotein(AFP), prostatespecific antigen(PSA), carcino-embryonic antigen(CEA) and mucin-1(MUC-1) were first simultaneously enriched from human plasma by aptamers immobilized capillary column with polymer@graphene oxide @polyethylenimine@Au(PM@GO@PEI@Au) modified microspheres as the matrix, followed by on-line protein denaturation, reduction, desalting and tryptic digestion by a fully automated sample treatment(FAST) device, finally the resulting peptides were analyzed by pararell reaction monitoring on a LTQ-orbitrap velos mass spectrometry. Compared to traditional ELISA assay widely used in routine clinical practice, such a platform exhibited significant advantages such as short analysis time( 2 h vs. 12 h), low limit of detection(for AFP : 5 fg/μL vs. 46 fg/μL, and for PSA : 50 fg/μL vs. 69 fg/μL), and ease of automation. Furthermore, our developed platform was also applied in the absolute quantification of tumor markers from clinical plasma samples, and the results were comparable to those obtained by clinical immunoassay. All the results demonstrated that our developed fully automated platform should provide a promising tool for achieving high sensitivity, high accuracy, and high throughput detection of tumor protein markers in the routine physical examination and clinical disease diagnosis.

关键词： Mass spectrometry absolute quantification tumor biomarkers human plasma

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High-throughput Absolute Quantification of Proteins by an Im...

quantification of CP4-EPSPS in genetically modified Nicotiana tabacum leaves by LC-MS/MS with ^(18)O-labeling

第十四届全国中药和天然药物学术研讨会

作者： Junying Wei Chen Ding Jiao Zhang Wei Mi Yan Zhao Mingwei Liu Tianyi Fu Yangjun Zhang Wantao Ying Yun Cai Jun Qin Xiaohong Qian

Stable isotope dilution-selective reaction monitoring-mass spectrometry（SID-SRM-MS） has been widely used for absolute quantitative analysis of ***,when performing large-scale absolute quantification of proteins from a more complex tissue sample,such as mouse liver,in addition to a high-throughput approach for preparation and calibration of large amounts of stable isotope-labelled internal standards,a more powerful separation method prior to SRM analysis is also urgently *** address these challenges,a high-throughput absolute quantification strategy based on improved two-dimensional reversed-phase（2D RP） separation and quantification concatamer（QconCAT） approach is presented in this *** strategy can achieve simultaneous quantification of hundreds of proteins from mouse liver with one week or less of total MS measurement *** using calibrated synthesised peptides from the glutathione S-transferase（GST） protein,large amounts of GST-tag QconCAT internal standards corresponding to hundreds of proteins can be accurately and rapidly ***,with the use of an improved 2D RP separation method,a mixture containing a digested sample and QconCAT standards can be efficiently separated and absolutely *** a maximum gradient of 72 min in the1 st LC dimension,resulting in 72 fractions,identification and absolute quantification experiments for all fractions can be completed within one week of total MS measurement *** quantification approach developed here can further extend the dynamic range and increase the analytical sensitivity of SRM analysis of complex tissue samples,thereby helping to increase the coverage of absolute quantification in a whole proteome.

关键词： Proteome absolute quantification Two Dimensional Reversed-phase Separation quantification Concatamer(QconCAT)

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Journal of Beijing Institute of Technology 2015年第2期24卷 277-284页

作者：张玫鄯科明徐伟林凡凯邓玉林 State Key Laboratory for Infectious Disease Prevention and Control National Institute of Communicable Diseases Control and Prevention Chinese Center for Disease Control and Prevention School of Life Science Beijing Institute of Technology Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases Planning and Research Institute Norinco Group

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 *** purification procedures.

关键词： CP4-EPSPS absolute quantification ^18O-labeling MRM

COMPOSITION DETERMINATION OF BINARY POLYMER MIXTURES BY SIZE EXCLUSION CHROMATOGRAPHY WITH LIGHT SCATTERING DETECTION

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Chinese Journal of Polymer Science 2010年第3期28卷 291-297页

作者：程镕时 Key Laboratory of Mesoscopic Chemistry of MOE College of Chemistry and Chemical Engineering Nanjing University College of Materials Science and Engineering South China University of Technology

Base on the principle of absolute quantification of size exclusion chromatography （SEC）, a light scattering （LS） detector coupled with a concentration detector （refractive index detector） is utilized to determine the compositions of complicated binary mixtures. A theoretical analysis predicts that the response factors for both LS and RI detectors are linear functions with the composition of any specified polymer mixtures in the binary polymer mixtures. Two pairs of complicated binary mixtures were used to test the theory mentioned in the present paper, and the experimental results show an excellent accordance with the theory.

关键词： Size exclusion chromatography Light scattering Composition of binary mixture absolute quantification Response factor.

Design and use of group-specific primers and probes for real-time quantitative PCR

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Frontiers of Environmental Science & Engineering 2011年第1期5卷 28-39页

作者： Juntaek LIM Seung Gu SHIN Seungyong LEE Seokhwan HWANG School of Environmental Science and Engineering Pohang University of Science and TechnologyPohangGyungbuk 790-784The Republic of Korea

Real-time quantitative polymerase chain reaction(qPCR)has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil,water,sediments,and *** qPCR is a very useful technique for nucleic acid quantification,accurately quantifying the target microbial group strongly depends on the quality of the primer and probe *** aspects of conducting qPCR assays have become increasingly routine and automated;however,one of the most important aspects,designing and selecting primer and probe sets,is often a somewhat arcane *** many cases,failed or non-specific amplification can be attributed to improperly designed primer-probe *** paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR *** demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal *** assays using group-specific primers and probes designed with this method,have been used to successfully quantify 16S ribosomal Ribonucleic Acid(16S rRNA)gene copy numbers from target methanogenic and ammoniaoxidizing bacteria in various laboratory-and full-scale biologic *** with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.

关键词： absolute quantification design guideline primer probe real-time quantitative polymerase chain reaction(qPCR)

Cell-free synthesis of stable isotope-labeled internal standards for targeted quantitative proteomics

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Synthetic and Systems Biotechnology 2018年第2期3卷 97-104页

作者： Ryohei Narumi Keiko Masuda Takeshi Tomonaga Jun Adachi Hiroki RUeda Yoshihiro Shimizu Laboratory of Proteome Research National Institutes of Biomedical InnovationHealth and Nutrition7-6-8Satio-AsagiIbarakiOsaka 567-0085Japan Laboratory for Single Cell Mass Spectrometry RIKEN Quantitative Biology Center(QBiC)6-2-3FuruedaiSuitaOsaka 565-0874Japan Department of Systems Pharmacology Graduate School of MedicineThe University of Tokyo7-3-1 HongoBunkyo-kuTokyo 113-0033Japan Laboratory for Synthetic Biology RIKEN Quantitative Biology Center(QBiC)6-2-3FuruedaiSuitaOsaka 565-0874Japan Laboratory for Cell-Free Protein Synthesis RIKEN Quantitative Biology Center(QBiC)6-2-3FuruedaiSuitaOsaka 565-0874Japan

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 *** high-sensitivity detection of lowabundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal ***,there are various ways for preparing standards,including chemical peptide synthesis,cellular protein expression,and cell-free protein or peptide ***-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 ***,we review the use of such systems for precise and reliable protein quantification.

关键词： absolute quantification Mass spectrometry Cell-free protein synthesis system In vitro translation Targeted quantitative proteomics PURE system

Designing specific bacterial 16S primers to sequence and quantitate plant endo-bacteriome

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Science China(Life Sciences) 2022年第5期65卷 1000-1013页

作者： Liying Chen Mengting Zhang Da Liu Hongbo Sun Jianxiang Wu Yan Huo Xiaoying Chen Rongxiang Fang Lili Zhang State Key Laboratory of Rice Biology Institute of BiotechnologyZhejiang UniversityHangzhou 310058China State Key Laboratory of Plant Genomics Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China National Plant Gene Research Center Beijing 100101China

Plant endophytic bacteria colonize the internal tissues of plants and interact with plants *** past two decades have witnessed the increasing application of next-generation 16S r RNA gene sequencing in the investigation of bacterial ***,deciphering plant endo-bacterial communities by this method is difficult because of the co-amplification of massive plant organellar DNAs with bacterial ***,we designed polymerase chain reaction(PCR) primer sets,including799F/1107R,322F/796R,and 322F-Dr/796Rs(primer pair 322F/796R with a penultimate-base substitution in 322F),that can specifically amplify bacterial 16S from plant total *** computationally and experimentally evaluated the specificity,coverage,and accuracy of the newly designed primer *** 799F/1107R and 322F-Dr/796Rs produced plant DNA-free 16S amplicon libraries or reduced plant DNA contamination to lower than 5% for the plant materials with extremely-low-abundance bacterial *** primer set 322F-A/796R was used through absolute quantitative PCR to quantitate the population size of rice leaf or root endo-bacteriome,which revealed 10^(6)–10^(7) and 10^(9)–10^(10) bacteria per gram fresh weight,*** 16S primer sets and amplification methods enable the simple and inexpensive next-generation sequencing and quantification of plant endo-bacteriome,which will significantly advance studies on the plant-related microbiome.

关键词： plant endo-bacteriome plant DNA-free amplification 16S rRNA gene next-generation sequencing absolute quantification

Quantitative analysis of glutamate compounds in the swine brain following central analgesics nasal spray using proton magnetic resonance spectroscopy and linear combination model

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Neural Regeneration Research 2009年第7期4卷 508-512页

作者： Jianlin Wu Ting Liu Yan Lin Zhiwei Shen Yanwei Miao Renhua Wu Changkai Sun Department of Radiology. First Affiliated Hospital Dalian Medical University Dalian 116011 Liaoning Province China Department of MedicaFimaging Second Affiliatecl Hospital Medical College Shantou University Shantou 515041 Guangdong ProvinceChina Devision of Neurophysiology Dalian Medical University Dalian 116023 Liaoning Province China

BACKGROUND： In localized brain proton magnetic resonance spectroscopy （^1H-MRS）, metabolite levels are often expressed as ratios, rather than absolute concentrations. Frequently, the denominator is creatine, which is assumed to be stable in normal, as well as many pathological, states. However, in vivo creatine levels do not remain constant. Therefore, absolute metabolite measurements, which provide the precise concentrations of certain chemical compounds, are superior to metabolite ratios for determining pathological and evolutional changes. OBJECTIVE： To investigate the feasibility of quantification analysis of brain metabolite changes caused by central analgesics nasal spray using the ^1H-MRS and linear combination model （LCModel） methods. DESIGN, TIME AND SETTING： This neuroimaging, observational, animal study was performed at the Laboratory of the Department of Medical Imaging, Second Affiliated Hospital, Medical College, Shantou University, China from July to December 2007. MATERIALS： Butorphanol tartrate nasal spray, as a mixed agonist-antagonist opioid analgesic, was purchased from Shanghai Hengrui Pharmacy, China. A General Electric Signa 1.5T System （General Electric Medical Systems, Milwaukee, WI, USA） and LCModel software （Stephen Provencher, Oakville, Ontario, Canada） were used in this study. METHODS： MRS images were acquired in ten healthy swine aged 2 weeks using single-voxel point-resolved spectroscopic sequence. A region of interest （2 cm × 2 cm × 2 cm） was placed in the image centers of maximum brain parenchyma. Repeated MRS scanning was performed 15-20 minutes after intranasal administration of 1 mg of butorphanol tartrate. Three settings of repetition time/echo time were selected before and after nasal spray administration 3 000 ms/30 ms,1 500 ms/30 ms, and 3 000 ms/50 ms. Metabolite concentrations were estimated by LCModel software. MAIN OUTCOME MEASURES： ^1H-MRS spectra was obtained using various repetition time/echo time settings. Co

关键词： proton magnetic resonance spectroscopy nasal spray linear combination model glutamate compounds absolute quantification

absolute quantitative detection of genetically modified soybean MON87708×MON89788 with stacked traits by digital polymerase chain reaction

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Oil Crop Science 2022年第4期7卷 180-188页

作者： Junyi Xu Xin Li Jinglian Bai Ying Liu Shaojie Wang Yueting Liu Chunguang Yang Technology Center of Dalian Customs District DalianChina Logistics Management Center of Dalian Customs District DalianChina

The main advantage of digital PCR(dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration *** droplet dPCR has a three-color staining detection function,which enables multiplex PCR *** this study,this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708 × MON89788 with stacked *** absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708× MON89788 *** results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.

关键词： absolute quantification Digital PCR Genetically modified soybean MON87708×MON89788 Stacked event