ITRAQ实验原理.ppt

ITRAQITRAQ实验原理简介实验原理简介Section 1:Section 1:实验原理实验原理Spectral counts In vitroIn vivoPeak area定量蛋白质组学技术定量蛋白质组学技术SILAC ICAT/iTRAQ/O18Isotope Tagging for Relative and Absolute protein Quantitation(iTRAQ)Can deferentially label and run up to Eight samples Proteins are digested prior to labeling Labels react with N-terminus No reduction of peptides based on amino acid compositionAnalyze all peptides Mass of peptide ions is the same allowing for a single mass to be selected for MS/MS Reporter group is lost during fragmentationUsed to determine relative abundance of selected peptide of interest from the four(or eight)samples iTRAQ reagent structureiTRAQ reagent structureiTRAQ reagent structureiTRAQ reagent structureiTRAQ LabelingiTRAQ LabelingiTRAQ LabelingiTRAQ LabelingiTRAQ PrincipleRoss,P.L.et al.(2004)Mol Cell Proteomics 3(12):1154-69iTRAQSample 1Sample 2Sample 3Sample 4Reduce&AlkylateTrypsin digestLabel w/114 tagLabel w/115 tagLabel w/116 tagLabel w/117 tagMix,dilute in SCX bufferSCXCapillary RPLC-MSRoss,P.L.et al.(2004)Mol Cell Proteomics 3(12):1154-69iTRAQRoss,P.L.et al.(2004)Mol Cell Proteomics 3(12):1154-69iTRAQiTRAQRoss,P.L.et al.(2004)Mol Cell Proteomics 3(12):1154-69-Peptides have the same mass from each of the samples-MS/MS of selected mass yields-Fragmentation spectra for the identification of peptide-Reporter group gives relative abundance informationSectioin 2:Sectioin 2:实验步骤实验步骤Experiment Design overview(eg)Experiment Design overview(eg)Sample preparation-1(eg)Sample preparation-1(eg)Sample preparation-1(eg)Sample preparation-1(eg)Preparation of Sample Pools.Potassium-EDTA plasma samples were collected by venipuncture from 15 human females.The plasma was collected,aliquoted,and stored frozen at-70 C.All study volunteers were 40 years of age and in generally good health.The study subjects were in two groups;eight presented as osteoarthritis(OA)patients and seven were agematched controls.OA patients were defined as having frequent knee symptoms in the past year,a Kellgren and Lawrence grades 2 or 3 on one or both knees,and a body mass index(BMI)g30.Age-matched control volunteers had no evidence of OA in the knee and a BMI 25)in the survey scan sequentially subjected to product ion analysis.4.Product ion spectra were accumulated for 2 s in the mass range m/z 100-2000 with a modified Enhance All mode Q2 transition setting favoring low mass ions so that the reporting iTRAQ ion(114,115,116 and 117 m/z)intensities were enhanced for quantitation.Dynamic exclusion was used with a 2 min and 150 ppm window.Data Analysis-1(eg)Data Analysis-1(eg)Data Analysis-1(eg)Data Analysis-1(eg)Peptide selection criteria for relative quantitationReplicate analysis and cutoff point estimationSignificant differentially regulated proteinProtein classificationValidation of iTRAQ identified Candidates合作客户网络图合作客户网络图合作客户网络图合作客户网络图项目合作客户分类项目合作客户分类项目合作客户分类项目合作客户分类合作的科研院校近合作的科研院校近200200家家:包括复旦大学、北京大学、上海交大、华东师大、军科院、中科院生物物理所、第四军医大学、广州中山大学中科院北京植物所、昆明动物所、华中农业大学、香港浸会大学等；临床医院近临床医院近150150家家:包括上海瑞金医院、上海中山医院、上海华山医院、上海第六人民医院、上海长海医院、上海长征医院、北京阜外医院、北京协和医院、北京301医院、南方医科、武汉同济医院 等;服务的生物医药公司超过服务的生物医药公司超过150150家家:其中包括:上海实业科华、华北制药、三九集团、珠海联邦、上海丽珠等生物制药公司;