Identifying Proteins with Tandem Mass Spectrometry Lectures识别和串联质谱蛋白质讲座ppt课件.ppt

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1、InterpretingMS/MSProteomicsResultsBrianC.SearleProteomeSoftwareInc.Portland,OregonUSABrian.SearleProteomeSoftwareNPCProgressMeeting(February2nd,2019)ThefirstthingIshouldsayisthatnoneofthematerialpresentedisoriginalresearchdoneatProteomeSoftwarebutwedostrivetomake

2、thetoolspresentedhereavailableinoursoftwareproductScaffold.Withthatcaveataside…IllustratedbyToniBoudreaultOrganizationThisisanforemostanintroductionsowe’refirstgoingtotalkaboutThenwe’regoingtotalkaboutthemotivationsbehindthedevelopmentofthefirstreallyusefulbioinf

3、ormaticstechniqueinourfield,SEQUEST.ThistechniquehasbeenextendedbytwoothertoolscalledX!TandemandMascot.We’realsogoingtotalkabouthowtheseprogramsdifferandhowwecanusethattoouradvantagebyconsideringthemsimultaneouslyusingprobabilities.IdentifySEQUESTX!Tandem/MascotD

4、ifferCombinehowyougoaboutidentifyingproteinswithtandemmassspectrometryinthefirstplaceSo,thisisproteomics,sowe’regoingtousetandemmassspectrometrytoidentifyproteins--hopefullymanyofthem,andhopefullyveryquickly.AAIKGKIDVCIVLLQHKAEPTIRNTDGRTAStartwithaproteinAndtouse

5、thistechniqueyougenerallyhavetolysetheproteinintopeptidesabout8to20aminoacidsinlengthand…AAIKGKIDVCIVLLQHKAEPTIRNTDGRTACutwithanenzymeAAIKGKIDVCIVLLQHKAEPTIRNTDGRTASelectapeptideLookateachpeptideindividually.Weselectthepeptidebymassusingthefirsthalfofthetandemmas

6、sspectrometerAEPTIRH2OImpartenergyincollisioncellThemassspectrometerimpartsenergyintothepeptidecausingittofragmentatthepeptidebondsbetweenaminoacids.M/zIntensityAEPAAEAEPT72.0201.1298.1399.2MeasuremassofdaughterionsThemassesofthesefragmentionsisrecordedusingthese

7、condmassspectrometer.M/zIntensityAEPTIRB-typeIonsH2O72.0129.097.0101.0113.1174.1TheseionsarecommonlycalledBions,basedonnomenclatureyoudon’treallywanttoknowabout…Butthemassdifferencebetweenthepeakscorrespondsdirectlytotheaminoacidsequence.M/zIntensityAEPTIRB-typeI

8、onsH2O72.0129.097.0101.0113.1174.1A-0AE-AAEP-AEAEPT-AEPAEPTI-AEPTAEPTIR-AEPTIForexample,theA-EpeakminustheApeakshouldproducethemassofE.Youcanbuildthesemassdiff