蛋白质组学

蛋白质组研究的技术策略与应用

蛋白质组研究的技术策略与应用
Development and Application of the Strategies on Proteome Research
钱小红
基因组(genome=gen+ome):生物体所拥有的全套染色体上的全部基因转录组(transcriptome=transcript+ome):一种细胞,组织,或生物体完整基因组所对应的全套mRNA
蛋白质组(proteome=prote+ome):一种细胞、组织或生物体完整基因组所对应的全套蛋白质
2001/2/15 The genome of human :Sequence of gene ( Nature )
2001/2/16 The genome of human :Function of gene ( Science )
30,000-40,000 genes in human genome sequence
The function of an estimated 1/3 unknown
The correlation between mRNA and protein levels is generally lower than 0.5
Proteomics
表达蛋白质组(Expression proteomics)Human Proteome Index Yeast Proteome Database (YPD,6145 proteins)
功能蛋白质组(Functional proteomics)Disease proteome Pharmaceutical proteome
Proteomics基本生物学问题
Post-translational modification
Protein-protein interaction
Genome-Transcriptome-Proteome
临床应用问题
Disease marker-diagnosis
Mechanism of the disease
Drug target-therapy
Proteomics
Profiling
Identification
Quantification
Localization
Modification
Interaction
Function
Three key techniques
2DE, 2D-HPLC---Separation
Computer Image Analysis---Analysis
Biological Mass Spectrometry--Identification
Protein separationTwo dimensional electrophoresis
IEFàequilibriumàSDS-PAGE1、合成载体两性电解质(SCA)
2、非平衡pH梯度电泳(NEPHGE)
3、固相pH梯度等电聚焦(IPG IEF)
1、线性SDS-PAGE
2、非线性SDS-PAGE
2D map of HL-60 cell treated with ATRA
2D-HPLC based proteomics Analysis Technique (shotgun)
Protein mixtureàPeptides pH<3à 2D-HPLC(SCXà RP) à Protein sequencing (Mass spectrometer)à Database searchà Protein identification
Image analysis
Silver staining gelàSpot detectàMatchingàDifferential mapàNormalizationàReportDifferentialy expressed proteins between cell lines
Protein identification
MALDI-TOF-MASS
Matrix Assisted Laser Desorption Ionization Time of Flight
Sample(Target)àIonization(Laser)à Separation(Flight Tube)à Detection(Detector)àComputer
生物质谱-功能基因组研究的关键技术之一
Sensitivity: fmol-amol Accuracy: 5-50ppm High throughput: 100个/小时
MALDI-TOF-TOF-MS
Protein sequencing (Tandem mass spectrometry)
Peptide mass fingerprinting
Mass accuracy <0.01%
Resolution > 10000
Sensitivity: fmol
Q-TOF (Q-STAR) Quadruple-Time of Flight (1997)
Sampleà Ionization(Ion source ESI)à MS1(Quadruple)à Mass selector(Quadruple)à MS2(Quadruple)à Separator and Detector(TOF)à Data processLC-ESI-MS/MSCapLC –nanospray
Mass accuracy <0.01%
Resolution > 10000
Sensitivity: fmol
Q-Tof Micro 蛋白质组研究整体技术的特点规模化
高通量
自动化
蛋白质组表达谱研究的技术要求高分辨率
高灵敏度蛋白质组研究的基本技术
计算机技术à分离/鉴定à……
2DE-MS/MS-MS 生产线 (一)2D-Gel分离系统,大规模染/脱色盘,摇床,扫描系统、切胶系统,自动酶切/点靶仪, 自动化串联测序系统, 自动切胶仪, 自动化酶切点靶系统, 自动化PMF鉴定系统
整合基础: BioRad公司和 Micromass公司的技术和约
2DE-MS/MS-MS生产线 (二)Ettan Spot Handling Workstation
Ettan MALDI Pro
Proteome X
2DLC-nano2DLC-MS/MS生产线
2D-LC for intact proteins
Gilson 215 collector
MALDI-TOF-TOF
Symbiot 自动点靶仪
2D Nano-LC for peptides
蛋白质组研究的技术策略(一)一维凝胶电泳-毛细管液相色谱-串联质谱 SDS-PAGE , LC-ESI-MS-MS
SDS-PAGE/MS-MS鉴定蛋白质策略
1.Sample fractionationàSDS-PAGEàExcised protein
2.Trypsin digestionàPeptide mixture
3.Peptide Chromatography and ESI
4.MS
5.MS/MS
SARS冠状病毒感染相关蛋白质组研究
军事医学科学院放射医学研究所
军事医学科学院微生物流行病研究所
军事医学科学院仪器测试分析中心
蛋白质组研究的技术策略(二)
蛋白全酶切-二维毛细管液相色谱-串联质谱 shotgun proteins peptides 2DLC-ESI-MS-MS
Shotgun Protein IdentificationProtein mixtureàProtein digestsàSCX clolumn fractionationàReverse column separationàMicro/Nano Electrospray Ionization on LCQ Ion TrapàAuto MS/MS detectionàTandem MS spectraàBioWorks database searchàResults
国际人类血浆蛋白质组计划(Human Plasma Proteome Project HPPP)
蛋白质组研究的技术策略( 三)
二维电泳-质谱2DE MALDI-TOF-MS MALDI-TOF-TOF-MS
蛋白质组研究的技术策略(四)二维色谱蛋白预分离-蛋白质酶切-二维毛细管液相色谱-串联质谱 Top-Down 2D-LC 2D-MicroLC-ESI-MS-MS proteins peptides
人胎肝蛋白质组表达谱的构建及其与转录组的比较
Human Proteome Organization HUPO 2001
促进国家与地区HUPO的成立与国际HUPO的发展
促进有关蛋白质组研究的国际学术交流与教育
协调公众蛋白质组合作研究HUPO 的五大合作研究领域2002. Washington
国际人类肝脏蛋白质组计划
Liver and Model Cell Proteomes HUPO Workshop Beijing, China October 22-24th, 2002
Jointly Organized by HUPO and Beijing Institute of Radiation Medicine
具体目标“两谱” ——表达谱、修饰谱
“两图” ——定位图、连锁图
“三库” ——样本库、抗体库、数据库
Thanks To:
Yongtao Yu Ying Jing Jian Wang Gang cheng Yunwei Hao Lihai Guo Rong Shi Songfeng Wu Ping Wan Jianqi Li Dong Li Yunping Zhu Wantao Ying Yangjun Zhang Jinglan Wang Xiaohai Li Jingquan Dai Shujia Dai Yun Cai Xiaohong QianACKNOWLEDGMENTSThis work was partially supported by:
Chinese National Key Project of Basic Research
Chinese National High-tech Program
Chinese National Distinguished Young Scholar Awards
Chinese National Natural Science Foundation Key Project
Beijing City Municipal Key Project
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