labrlfree非标记定量蛋白质组学的分析方法x

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

2、grequiredCompatiblewithvirtuallyanysampleSpectralCounting#MS/MSidentifiedforeachproteinVerycrudeEasilyimplementedSomewhatsampledependentSpectralcountsaresometimesnormalizedacrossmassspecrunsExtractedIonChromatogram(XIC)SumofareasunderthecurvesofMS1chromatogramsofeachpeptideidentifiedRequiresadvanc

3、edprocessingsoftwareSomewhatindependentofthesampleLabelFreeQuantitationinMaxQuantSILACratiocalculationrequiresthecomparisonofXICsbetweenthelabelstatesinamassspecrunCanusesameprocessingtocalculateXICsofpeptidesineachrunandthencompareacrossmassspecrunsNeedtomapfromonemassspecruntoothersEasyifMS2iden

4、tifiedinbothrunsHarderifnotidentifiedinbothrunsMaxQuanthasoptiontomatchidentifiedpeakstounidentifiedpeakusingretentiontimeandaccuratemassLFQOptionsinMaxQuantPeptideLevelRawintensityonlyoptionProteinLevelRawintensityMS2countsLFQvalueNopublicationyetCalculateglobalrawfilenormalizationfactoriBAQNorma

5、lizeintensitiesbynumberofobservablepeptidesBenchmarkingLFQOptionsTriplicateyeastanalysesPearsonCorrelationTypicalcorrelationmeasureSensitivetooutliersSpearmanRankCorrelationOnlylooksatrankingInsensitivetooutliersRatio95%ConfidenceIntervalTellsusthetypicalrangeofratioswewouldexpecttoseeforvaluestha

6、tareactuallyunchangedPeptideLFQPerformanceProteinRawIntensityLFQPerformanceProteinSpectralCountsLFQPerformanceProteiniBAQLFQPerformanceProteinLFQPerformanceLFQSummaryNowpossibletogetreasonableestimatesofrelativeabundancesbetweensamplesatboththepeptideandproteinlevelProtein95%CIat~1.5foldchangefor1

7、:1Peptide95%CIat~3foldchangefor1:1MightconsiderusingthesetechniquestomeasurelargechangesinmodificationstatusForexample,proteinfootprintingUbiquitinModificationDataMiningUbiquitinmodificationsresultinadiGlycinemod