labrlfree非标记定量蛋白质组学的分析方法.pptx

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1、IntroductionLabelFreeQuantitationinMaxQuantGlyGlyDiagnosticPeaksNewAnnotationVisualizationOptionsLabelFreeQuantitation(LFQ)Comparequantitativemeasuresofproteins/peptidesBETWEENmassspecrunstoidentifysignificantchangesComparisonisWITHINthemassspecrunforSILAC,iTRAQ,andIPTLNoche

2、micalormetaboliclabelingrequiredCompatiblewithvirtuallyanysampleSpectralCounting#MS/MSidentifiedforeachproteinVerycrudeEasilyimplementedSomewhatsampledependentSpectralcountsaresometimesnormalizedacrossmassspecrunsExtractedIonChromatogram(XIC)SumofareasunderthecurvesofMS1chro

3、matogramsofeachpeptideidentifiedRequiresadvancedprocessingsoftwareSomewhatindependentofthesampleLabelFreeQuantitationinMaxQuantSILACratiocalculationrequiresthecomparisonofXICsbetweenthelabelstatesinamassspecrunCanusesameprocessingtocalculateXICsofpeptidesineachrunandthencomp

4、areacrossmassspecrunsNeedtomapfromonemassspecruntoothersEasyifMS2identifiedinbothrunsHarderifnotidentifiedinbothrunsMaxQuanthasoptiontomatchidentifiedpeakstounidentifiedpeakusingretentiontimeandaccuratemassLFQOptionsinMaxQuantPeptideLevelRawintensityonlyoptionProteinLevelRaw

5、intensityMS2countsLFQvalueNopublicationyetCalculateglobalrawfilenormalizationfactoriBAQNormalizeintensitiesbynumberofobservablepeptidesBenchmarkingLFQOptionsTriplicateyeastanalysesPearsonCorrelationTypicalcorrelationmeasureSensitivetooutliersSpearmanRankCorrelationOnlylooksa

6、trankingInsensitivetooutliersRatio95%ConfidenceIntervalTellsusthetypicalrangeofratioswewouldexpecttoseeforvaluesthatareactuallyunchangedPeptideLFQPerformanceProteinRawIntensityLFQPerformanceProteinSpectralCountsLFQPerformanceProteiniBAQLFQPerformanceProteinLFQPerformanceLFQS

7、ummaryNowpossibletogetreasonableestimatesofrelativeabundancesbetweensamplesatboththepeptideandproteinlevelProtein95%CIat~1.5foldchangefor1:1Peptide95%CIat~3foldchangefor1:1MightconsiderusingthesetechniquestomeasurelargechangesinmodificationstatusForexample,proteinfootprintin

8、gUbiquitinModificationDataMiningUbiquitinmodificationsresultinadiGlycinemod