phosphorylation site mapping- maximizing the outcome

PhosphorylationSiteMapping-maximizingtheoutcome

PresentedbyHenrietteA.Remmer,Ph.D.

Director,BRCFProteomics&PeptideSynthesisCoreResearchAssistantScientist,Dept.ofBiologicalChemistry

PROTEOMICS & PEPTIDE SYNTHESIS CORE

Phosphoproteomics

• PhosphorylationonSer,Thr andTyristheprevalentPTMandoccursonover30%ofthehumanproteome.

• Thehumangenomeencodesfor518proteinkinasesandapprox 150proteinphosphatases• PhosphorylationoccursmainlyonSer residues(86%),followedbyThr residues(12%)

andTyrresidues(2%).• Phosphorylatedpeptidesusuallyhaveanabundanceoflessthan5%,thereforeselective

enrichmentisrecommendedpriortoanalysis.• Localizephosphorylationsitesonindividualproteins• Localizephosphorylationsitesonproteomestoanalyzeinformationflux

throughsignalingpathways

ü Phosphoproteomics workflowü LocalizationofphosphorylationsitesusingScaffoldsoftwareandmanualverificationü Parameterstovaryinordertomaximizenumberofphosphorylationsites

SAMPLESIn-geldigestion

Phospho-peptideenrichment

MassSpectrometryLC-MS/MS

DATAANALYSIS

Phosphoproteomics Workflow

Coomassiestainedgelbands

Reductionandalkylationfollowedbytryptic digest:CarbamidomethylCys

•TiO2 tip•Elutionwith5%NH4OH(inwater)and5%pyrrolidone(inwater)

• SeparationofpeptidesbyLC

• MSofpeptide(parention)

• MS/MSofpeptidetodeterminefragmentions/peptidesequence

• Databasesearch(proteinIDandphosphorylationasvariablemodificationonS,T,Y)

• Softwareused:MascotScaffoldScaffoldPTM

• Manualspectrumreviewtoverifymodification

Proteins

Peptides

Massspectrometryanalysis:

TheProcessofProteomicAnalysis Proteolytic Cleavage

Mascot: ionscore>45proteinscore>78

X!Tandem:-logEvalue<0.01Scaffold: (peptideprophet)

probability>95%

Dataanalysisandinterpretationbeginswithadatabasesearch

• TheexperimentallyobtainedMS/MSspectraofthepeptidesarematchedagainsttheoreticalMS/MSspectraavailableinthepublicdatabases(NCBIand/orUniprot)

• Completeorpartialpeptidesequencesareassignedandproteinsareidentifiedviatheiruniquepeptides

• Searchenginesareusedintheprocess,usingathresholdbasedscoringtodistinguishpositive(correct)peptideidentifications.

• Scaffoldusesprobabilitybasedscoring.NCBI=NationalCenterforBiotechnologyInformation;http://www.ncbi.nlm.nih.gov/Uniprot Knowledgebase:www.expasy.org

• Scaffoldisasoftwarethatdisplaysthedataandresultsfromproteinidentification andqualitativeandquantitativeLC-MS/MSanalysis

• Scaffoldhasa“freeviewer”versionfordatathatuserscandownloadfromwww.proteomesoftware.com.

• Scaffoldalsoallowstheusertoidentify,compareandquantitateproteins,toreviewandvalidateMS/MSspectraandsequenceassignmentsincludingthoserelatedtoposttranslationalmodifications(localizePTMs).

• Scaffold’sscoringisbasedonthePeptideProphet*Algorithm.• Probabilitybasedscoringallowsforeasydatainterpretation

andcomparabilitybetweenexperiments.Ithasbeenverifiedinmany,manysamples.

What is Scaffold?

*A.Keller,A.Nesvizhskii,A.Kolker,R.Aebersold (2002),Anal.Chem.74,5383-5392

Scaffolddisplaysthelistofidentifiedproteins(non-redundantproteinlist)andprovidesalotofadditionalinformationtosupporttheID.

DataOutputfromaproteinIDexperimentinScaffold

DataOutputfromaproteinID/GeLCMS experimentinScaffold

TheProteinViewshowsthesequencecoveragemapforeachproteinandforeachsample


TheProteinViewalsoshowstheMS/MSdataforeachidentifiedpeptideidentifiedaswellasposttranslationalmodificationspresent


Thefragmentationtabledisplaysthefragment–ionsobserved.

PeptideFragmentationbyMS/MS-(CID)

(N-terminus)Asp------Gly-----------Gly---------Lys(C-terminus)1234

Peptidesfragmentatthepeptidebondandyieldtwoion–series:Theb-ions containtheN-terminusofthepeptide–theyarenumberedfromlefttorightThey-ionscontaintheC-terminusofthepeptide–theyarenumberedfromrighttoleft

Asp--------Gly--------------Gly-----------Lys

b1 b2 b3

y3 y2 y1

InScaffold,b-ions aredisplayedinred,y-ions aredisplayedinblue.

LocalizationofPTMs

–ThemassofthePTMisincludedintheMascotdatabasesearch.ThemascotfilesareuploadedintoScaffold.ThesitesofmodificationarereportedinScaffold.

–Manualspectrumreviewisnecessarytoverifymodifiedpeptides.

–ScaffoldPTMisasoftwarepackagethatassistsinverificationoflocalizationofPTMs.Itisespeciallyrecommendedforphosphorylationsitemapping

LocalizationofphosphorylationsitesinScaffoldPTM

Localizingphosphorylationsitesinpeptides:theA-Score

• TheAmbiguityScore(A-Score)measurestheprobabilityofcorrectphosphorylationsitelocationwithinapeptidebasedonpresenceandintensityofsitedeterminingpeaksintheMS/MSspectrum

• TheAscoreiscalculatedasthedifferencebetweentheprobabilisticscoresofthetoptwopossiblesites

• Thealgorithmusesapeakdepthofupto10peaksper100m/z

• A-Scoresof>13aresignificant

Thepeptidescoretabprovidesvisualconfirmationofamodification’slikelihoodononeaminoacidovertheotherpossibleoptionsinthepeptide.


AnalysisofphosphorylationsitesontheQ-Exactive instrument,withenrichmentandCID

SequenceCoverageMap(33%sequencecoverage):

7confirmedPhosphorylationsites:T156,S159,S360,S377,S431,S664,S666.

1phospho moietypresent,butexactsiteNOTconfirmed:S441andS545

AnalysisofphosphorylationsitesontheQ-Exactive instrument,withenrichmentandCID


7confirmedphosphorylationsites:Confirmed:T156,S159,S360,S377,S431,S664,S666.

1phospho moietypresent,butexactsiteNOTconfirmed:S441andS545

T156:Spectrumandfragmentationtable:phosphorylationsitedirectlyconfirmedbyb-ionseries

S360:Spectrumandfragmentationtable:phosphorylationindirectlyconfirmedbypeptidemass.FragmentionsshowthatotherSer/Thr sitesarenotphosphorylated.

ScaffoldPTMresultsforphosphorylationsiteS360

A-Score

S545: phospho moietypresent(byparentionmass),exactlocalizationofthatsitenotconfirmedduetopoorfragmentationofthislongpeptide.

S545

Scaffold PTMresultsforphosphorylationsiteS545

AnalysisofphosphorylationsitesontheFUSIONinstrument,withenrichmentandCID


17confirmedPhosphorylationsites:S28,S30,T63,S142,T156,S159,S309,S354,S360,S377,S431,S441,S545,S559,S664,S666Notconfirmed:S418,S448,T449,S450,S451,T535,Y544,T547,S551,S553

S545:confirmedphosphorylationsite

ObtainedsignificantlymoreMS/MSspectra,Amongthemafewwithmuchbetterpeptidefragmentationthatallowtoconfidentlyassignthephosphorylationsite

PeptideFragmentationbyMS/MS-(ETD)

(N-terminus)Asp------Gly-----------Gly---------Lys(C-terminus)1234

Peptidesfragmentatthepeptidebondandyieldtwoion–series:Thec-ions containtheN-terminusofthepeptide–theyarenumberedfromlefttorightThez-ions containtheC-terminusofthepeptide–theyarenumberedfromrighttoleft

Asp--------Gly--------------Gly-----------Lys

c1 c2 c3

z3 z2 z1

ETD=ElectronTransferDissociation,(peptidebackbonedissociatesbetweenC-alphaandN-alpha)producingcandzions,aminoacidsidechainstaysintact,thereforeusefulforphosphorylationsitemapping

• 1confirmedphosphorylationsite:S75.• SevenpossiblephosphorylationsitespresentintheC-terminalpeptide,• 2phospho moietiespresentasconfirmedbyparentionmass• Exactlocationofthesetwositesnotconfirmedbyfragmentspectra

AnalysisofphosphorylationsitesontheQ-Exactive instrument,withenrichment,CIDandtryptic digest

Sequencecoverage:19%

ThepowerofETD

AnalysisofphosphorylationsitesontheQ-Exactive instrument,withenrichment,ETDandGlu-Cdigest

11Phosphorylationsitesunambiguouslyconfirmed:S75,S80,T84,S87,S97,S100,S101,S102,S150,S196 andS198.Phosphorylationlikelybutnotunambigously confirmed:S129.

ThepowerofETD

Sequencecoverage:67%

SummaryandConclusion• TheOrbitrap FusionLumos instrumentwithitsincreasedsensitivityallowsfor

morephosphorylationsitestobedetectedandassignedwithconfidence.

• Mascot/ScaffoldandScaffoldPTMareprovenandefficienttoolstohelpassignphosphorylationsiteswithconfidence.

• Phosphorylatedpeptidesusuallyhaveanabundanceoflessthan5%,thereforeselectiveenrichmentisrecommendedpriortoanalysis.

• Usingdifferentproteasesand/orcollisionmodes(CID/ETD)canenabletheconfidentassignmentofmorephosphorylationsites

• Startwithsufficientprotein

• Thecombinationofallthesefactorsyieldsthebestpossibleprojectoutcome

PROTEOMICS & PEPTIDE SYNTHESIS CORE

phosphorylation site mapping- maximizing the outcome

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