ITRAQ实验原理和定量蛋白质组学简介.

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2、lotfs21oe«1-2 Jen2 loss2-3 logsLsbel2-3 ion(spectnun couuuugjSpectral countsSILACuTiole prottwne AnalysisQVAQdfanv LneaiAccunrve Profeoine D>niamKProcess)C overdueRaB£eucnipicx Diocaemiaii umtiKw?Wtfabohe proteifl labekog CompAfnon of 2-3 代Cell cuitute wMctm onlyChemical pepude bbeliog

3、Xfecwim cotnplexin* bicbenttcal worialw?(MS)Compuiwn of 2-3 statesCbenuc pepude bbeliog Mnuuoi camplmrv biachMmol wonoloft i(MS MS)CompMKon of 2-8 ttateiMeaium comiEtv UcxnciiiKal workOon,EnzMtutic UbeuAgiMSlCompaiiwu or 2 stalerGel-slauiiiig ijilensity basedMass spectrometry basedSpiked peptidesLab

4、el fireefion mrensttv)I,al)el-ireeICAT/iTRAQ/018« 1. »种质迫*方扶的购性廁应用«K1lt较【231Medium commeuiv biocaauical workflowsTanked anatv&n of few protemsSimple bicchenucal woikflon-sCompanion of uiultiule statesSimple biochemical workflowsuTiole proteonie aoalvsisCompansofl of oiultipJe stat

5、esIsolope Tagging for Relative and Absolute prolein Quantitation(订 RAQ)Can deferentially label and run up to Eight samplesProteins are digested prior to labelingLabels react with N-terminusNo reduction of peptides based on amino acid compositionAn alyze all pep tidesMass of peptide ions is the same

6、allowing for a single mass to be selected for MS/MSReporter group is lost during fragmentationUsed to determine relative abuncfanee of selected peptide of interest from the four (or eight) samplesh12NtrypsinolysisH2NCoNoorKH2N 口 lamp*or 八.rIrckclionalton t -1 I打;UQ LahaJhJdlabelingJ. 一寸 |BatanceNRep

7、orter ionsmfzVb- and y-lons with additive Intensitiesr -r 二ReporterBalance100%m = 28Condition 2m = 114N HRoporter|I_| Balancem = 31iTRAQ principiePrinci pieedition 1 1J Extract2) Digest3) LabelRepresentative MS spectrum generated using iTRAQRepresentative MS spectrum generated using iTRAQpepTioeDI O

8、 IQE3b而1MixM5/MS0MS0伽l-WH. »EP TIDE-Peptld* fraflmvnta EQUAL eporlBT lorn DIFFERENTAniine spec peptde reactrvv group (hMS)111)- )0 j-MMg 4 PCPTlOe114Pl 11 -WMS 4 PCPTlOCny-j 3b |-WM« + ” TIDE 4Balanot Group Wan 31-2B (Meum lews)Refrortor Balanc* Pvptkto INTACT -4 sampieB kM/idca, mal>ot

9、aric Tag ToW Flaw 人_Rworttr G/OU> mM«1M -IV (R«<9>rs Chrve) A F8-PEPTIDEYlOi 'H-FBPTIDIYL咼114 (卄"3mfi iie <*n"Cio<«rvtfz IK (*3)«Ctt*nmft 117 (7gPEPTID*Ross, R L et al. (2004) Mol Cell Proteomics 3(12): 1154-69Sample 1Reduce &【rypsin Label w/Alkyf

10、ate digest 114 tagSample 2Sample 3Sample 4Mix, dilute in sex bufferLabel w/ 115ta A=Label w/116A 二Capillary RPLC-MSLabel w/Ross, R L et aL (2004) Mol Cell Proteomics 3(12): 1154-69iTRAQ1 fs£_r1352.64Mrs rrVxlSrUS 二-9P1110112.»114-61”4U»« (nMz)iii)"44、*iv)122 120.<r57 7M 7

11、61 g7*7 M9 C71573«75077”9Itau (mz|M“ (nWz)Ross, R L. et al. (2004) Mol Cell Proteomics 3(12): 1154-69iTRAQ1:1:1:1 Mixture1:5:2:10 MixtureuS"1.0"“MwaAti) ” -Pep tides have the same mass from each of the sam pies -MS/MS of selected mass yields-Fragmentation spectra for the ide ntificati

12、on of pep tide -Reporter group gives relative abundance informationRoss, R L. et ak (2004) Mol Cell Proteomics 3(12); 1154-69Sectioi2：实验步骤Experiment Design ovehrt帥(尊矢Serum 2Serum 3Serum 4Serum 1ITKAQ lbvl11 KAQ rntwlrr>p«)iiliQvttitunrxlrBctlon aI Huh nnundunlrikiracllun aI Hull ununaanlTrvp

13、UnDlfHTAlionI ryp*inpM ractlon ae11 lull ahundunlK-Alh 4l<-|frl«*ll4rJl1 ciMlneM radiun aI llvh ul>undanlk'lafe 4l<-ifrlL-lJiiiiITKAQ rnbri114ITKAQ rnbrlnsMix(>fT*llne Sunipic C'lean-up & Pcpildc Fructlonntlun Mith SCX HPI-V Column nay IlsPrep aration of Sam pie Po ols.P

14、otossium-EDTA plasma sampics were collected by venipuncture from 15 human females.The plasma was collected, oliquoted, 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 os osteoorthritis (

15、OA) paTicirrs and seven were agcmoTched controls. OA poTienTs were defined as having frequent knee symptoms in the post year, a Kcllgrcn and Lawrence grades 2 or 3 on one or both knees, and a body mass index (BMI) g30,Agc-matchcd control volunteers hod no evidence of OA in the knee ond a 8M1 <28-

16、Pooled scrum samples were prepored prior to immunodepleYion by combining 50 比 of plasma from individuoi samples (Table 1) Pool 1 was pre pared from 6 controls (C) and 6 patient samples (D) Pool 2 was a subset of Pool 1, containing 3 controls and 3 patierrt samples. Pool 3 was formed from a differert

17、 subset of Pool 1 using 3 controls and 3 p otic nt sam pies that were different from those used for Pool 2 Samples C7. D7, and D8 were not added to any poolDepletion of High-Abundance Proteins.Crude sera were thawed, diluted five-fold with buffer A (product no. 5185-5987, pH 7,4; Agilent Technologie

18、s, Palo Alto, CA, USA) and then filtered through 0.22 mm f liters (Agilent Technologies) by spinning at 16 000 6 gat room tempererfure for 15 min.Diluted scrum samples were injected on a Multiple Affinity Removal System HPLC column (Agilent Technologies) in 100% buffer A at a flow rate of 0,25 mL/mi

19、n for 9 min.The bound proteins were eluted in 100% buffer 6 at a flow rate of LO mL/min for 3.5 min. All chromatographic fractionations were performed at room temperature (20° C) on an HPllOO HPLC system with automated sample injector set at 4° C.The unbound (low-abundant) ond bound (high-

20、abundant) proteins were collected into Eppendorf tubes and stored at *20° C for further analysis.Acetone P recipitatSon1. Chill acetone to -20 ° C and the sample tube containing the sample to 4 * C.2 Add SIX volumes of cold acetone to the cold sample tube.3. Invert the tube three times.4.I

21、ncubate the tube at -20 ° C until prccipitotc forms (30 minutes to four hours).5. Decant the acetone.6. Use the precipitated pellet as your somple in "'Reducing thcProtcins and Blocking Cysteines/. Reducing the Proteins and Blocking Cystc/ncs1. To each o sampie tube containing 5 to 100

22、 pg of sampie (or thegr稈pifated pellet from acetone prccipi卞afion), odd 20 pL Dissolution 2 Add 1 pL of the Denaturant in the kit and vortex to mix. 3 To each sample tube, add 2 pL Reducing Reagent.4. Vortex to mix, then spin.5. Incubate the tubes at 60 C for 1 hour.6. Spin to bring the sample to th

23、e bottom of the tube.7. To each tube, add 1 pL Cystcinc-Blocking Reagent.8. Vortex to mix, then spin.9. Incubate the tubes at room tempcraturc for 10 minutes.Tryptic Digestion and iTRAQ Label!ngA total of 100 fjg of protein was digested overright w汁h trypsin at 37 ° C at a rotio of 1:20, trypsi

24、n to protcin.Digested sampies were labeled with the iTRAQ rcaqcnts following the protocol provided by tnc vendor (Applied Biosystems, Foster city, CAj. Briefly, one vial of iTRAQ labeling reagent was used for every 100 幻 of protcin,Ethonol was used to solubilize the iTRAQ reagent then added to the p

25、eptide sample ensuring a final organic concentration of at least 60% (v/v).After 1 h of iTRAQ labeling the react ion wo quenched by adding 50 /L of water. The sompies were then mixed at equal ratios and dried by centrifugal evaporation.Label exp criment design:Strong Cation Exchange Chromatography.i

26、TRAQ labeled pep tides were fractionated by strong cation exchange liquid chromatography (SCX) using C18 column： (Buffer A was 5 mAA phosphate 25% (v/v) acetonitrile, pH 2,7, and buffer B was 5 mM phosphate. 350 mM potassium chloride, 25% (v/v) acetonitrile, pH 2.7.)The dried iTRAQ labeled sample wa

27、s resuspended in buffer A and applied to the sex column.After sample loading and washing with buffer A for 37.5 min, buffer B concentration increased from 10% to 60% in 46 rrin and then romped to 100% at o flow rate of 200 /jL/min, Fifteen (15) fractions were collected from the SCX separation then dried by ccntrifugol evaporation. Reverse Phase NanoLC ESI MS/MS.1. Dried sex fractions were solubilized in 100 pL of 01% (v/v) formic acid,2% (v/v) acetonitrile.2. The LC eluen