Fusionfusioncharts

BAmericanSocietyforMassSpectrometry,2011DOI:10.
1007/s13361-011-0234-5APPLICATIONNOTEHDXFinder:AutomatedAnalysisandDataReportingofDeuterium/HydrogenExchangeMassSpectrometryDannyE.
Miller,CharulataB.
Prasannan,MariaT.
Villar,AronW.
Fenton,AntonioArtiguesBiochemistryandMolecularBiology,TheUniversityofKansasMedicalCenter,MS3030,3901RainbowBlvd.
,KansasCity,KS66160,USAAbstractHydrogen/deuteriumexchangeincombinationwithmassspectrometry(H/DMS)isasensitivetechniquefordetectionofchangesinproteinconformationanddynamics.
However,wideapplicationofH/DMShasbeenhindered,inpart,bythelackofcomputationaltoolsnecessaryforefficientanalysisofthelargedatasetsassociatedwiththistechnique.
Wereportanovelweb-basedapplicationforautomaticanalysisofH/DMSexperimentaldata.
Thisapplicationreliesonthehighresolutionofmassspectrometerstoextractallisotopicenvelopesbeforecorrelatingtheseenvelopeswithindividualpeptides.
Althoughafullyautomaticanalysisispossible,avarietyofgraphicaltoolsareincludedtoaidintheverificationofcorrelationsandrankingsoftheisotopicpeptideenvelopes.
Asademonstration,therateconstantsforH/Dexchangeofpeptidesfromrabbitmusclepyruvatekinasearemappedontothestructureofthisprotein.
Keywords:Hydrogen/deuteriumexchange,Massspectrometry,Proteindynamics,FouriertransformioncyclotronmassspectrometryIntroductionThereisagrowingappreciationthathydrogen/deuteriumexchangemassspectrometry(H/DMS)canprovideinsightsintodifferentaspectsofproteinconformationanddynamics.
Thus,thereisacurrentneedforcomputationaltoolsthatcanaidintheanalysisoftheverylargedatasetsproducedbythesetechniques.
Effortsinprogramdevelopmentto-datefocusonisolatedaspectsofdataanalysis;examplesincludecentroidcalculation[1,2],calculationofdeuteriumcontentinproteinsorpepticpeptides[3],anddeterminingenvelopedata[4].
Thesetoolsareusefulforthestatisticalanalysis,visualization,andplottingofdeuteriumcontent.
Thereareseveralproblemsassociatedwithcomplexmulti-dimensionalH/DMSexperimentsthathavenotbeenfullyaddressedbytheprogramslistedabove.
Oneisthedetectionofpeptideionisotopicenvelopes,complicatedbytheshiftinmassandcomplexityasaconsequenceofvaryingdeuteriumcontent.
Anotheristheisolationofindividualisotopeenvelopesfromoverlappingpeptideions.
ProgramssuchasHydra[5],HeXicon[6],theDeuterator[7],anditssuccessor,HDDesktop[8]attempttoaddresstheseproblemsusingstatisticalmethodsforfittingexperimentaldatatotheoreticalisotopeenvelopes.
Unfortunately,theseprogramsrequire,Received:24March2011Revised:3August2011Accepted:12August2011ElectronicsupplementarymaterialTheonlineversionofthisarticle(doi:10.
1007/s13361-011-0234-5)containssupplementarymaterial,whichisavailabletoauthorizedusers.
Correspondenceto:AronW.
Fenton;e-mail:afenton@kumc.
edu,AntonioArtigues;e-mail:aartigues@kumc.
eduJ.
Am.
Soc.
MassSpectrom.
(2012)23:425Y429PublishedOnline:15November2011asinitialinput,theHPLCretentiontimesofthepeptideionsofinterest.
Thus,evenwiththeseaids,userinputisneededatallphasesofdatainterpretation,makingthisprocesstime-consumingandpronetohumanerror.
TherearealsorecentdevelopmentsintheanalysisofH/DMSdataacquiredona14.
5TeslaFT-ICRinstru-ment[9],buttheseinstrumentsarenotcommonlyavailable.
HerewereportHDXFinder,anapplicationforpeptidedetectionandforautomaticpeakassignments,whichdoesnotdependonHPLCretentiontimes.
Detectionandisolationofisotopepeptideenvelopes,includingextrac-tionofoverlappingpeptideions,arefullyautomaticfunctionsperformedbyHDXFinder.
ThesetoftoolsincludedinHDXFinderhasbeentestedataresolutionpoweraslowas12,500foranionwithm/z=400.
ProgramDescriptionandResultsProgrammingBackgroundHDXFinderisaLinux-basedwebapplicationaccessiblefromanywebbrowser.
TheprogramisacombinationofJava,JavaScript,andPerl.
TheprogramutilizesaMySQLdatabaseforstorageandretrievalofalldata.
MostsoftwareusedinthedevelopmentandoperationofHDXFinderisfreelyavailableasopen-sourcesoftware.
TheonlyexceptionistheFusionChartsv.
3.
0packageusedtodevelopmostofthegraphicsfordataanalysis.
HDXFinderisfreelyaccessiblefromtheKUMedicalCenterMassSpectrometryCoreLaboratory(https://HDXAnalyzer.
kumc.
edu/)toallnot-for-profitusers.
Duetotherecentuseofthename"HDX-Analyzer"byanothergroup,wehavemodifiedthenameoftheprogramdescribedherefrom"HDXAnalyzer"to"HDXFinder.
"Therefore,theaddressoftheprogram(https://HDXAnalyzer.
kumc.
edu/)doesnotmatchtheprogramname(HDXFinder).
DataInputandAnalysisThreesetsofdataarerequiredasinputfortheprogram:(1)adatafile(s)containingthemassspectrometricinformation,(2)thesequenceoftheproteinofinterest,and(3)alistofpeptidesgeneratedfromtheproteinofinterest.
Additionally,apdbdatafilecanbeuploadedforrepresentationoftheoutputdataonthethree-dimensionalstructureoftheprotein.
HDXFindercanbeusedfordataanalysisoffilesgeneratedbyavarietyofinstruments,independentofmanufacturer.
Thus,itacceptsdatainthemzXML[10]ormzML[11]format,bothdevelopedtofacilitateexchangeofmassspectrometricinformationacrossmultipleplatforms.
Therearefreelyavailableconvertersforthespecificdatafilesgeneratedbymostmanufacturersofmassspectrometers(http://tools.
proteomecenter.
org)[12].
Thelistofpeptidesofinterestcanbegeneratedfromanyofthesearchenginesbroadlyusedforproteinidentification(atopicbeyondthescopeofthisarticle).
Foreachpeptideincludedinthislist,thefollowinginformationmustbeincludedinacomma-separated(csv)format:thepeptidesequence,thepeptidemass(M+H+),andthepeptidecharge.
Fortheexperimentsdescribedhere,thelistofpeptidesofinterestfromthepepticdigestofrabbitmusclepyruvatekinasewasgeneratedfromSequest(seeSupplementaryinformation).
ThebasiccomponentsandstepsoftheanalysisofafullH/DMSexperimentareoutlinedinFigure1.
ThechartshowsaschematicoftheworkflowofHDXFinder.
UponinputofmzXMLormzMLdata,theprogramautomaticallybeginsaseriesofconsecutivealgorithmstodetectandisolateisotopicenvelopes,measurethechargesoftheseenvelopes,andcalculatetheaveragemassoftherepresentedions.
AlldataarestoredinaMySQLdatabaseandcanbeobtainedinacsvformat.
HDXFinderwillmatch(i.
e.
,identifyenvelopesthatcouldcorrespondtopeptideionswithFigure1.
HDXFinderworkflow.
Thefinaloutputisatable,whichmaybeexportedasacsvfilecontaining,foreachpeptideandexperimentaltime(orcondition),thepeptide'sHPLCretentiontime,averagemass,thenumberofdeuteriumacquiredandoverallrateofexchange.
Finally,ifapdbfileisassociatedwiththeproject,thedeuteriumcontentorratescanbedisplayedonathree-dimensionalimageoftheproteinD.
E.
Miller,etal.
:AutomatedAnalysisofH/DExchangeMassSpectrometry426arangeofdeuterationcontent,dependingonthenumberofexchangeableamideprotons),correlate(i.
e.
,assignisotopicenvelopestoindividualpeptides),andrankthisdata(i.
e.
,associatesisotopicenvelopesacrossdifferentH/Dreactiontimes)(seeSupplementarymaterialfordetails).
Theuserhastheoptiontovalidatethesecorrelationsandrankingsand/orchangetheranking.
Ifdesired,rateconstantsforH/Dexchangecanbecalculatedandmappedontotheuserprovidedpdbstructure.
DeterminingtheRateConstantsofExchangeDeuteriumcontentismeasuredasthedifferenceofaveragemassbetweenthedeuteratedandthetheoreticalmassoftheundeuteratedpeptide.
Oncedatahavebeencorrelatedandranked,rateconstantsofexchangecanbecalculatedforeachpeptide.
Rateconstantsofexchangearedeterminedbyfollowingthemassofthepeptideasafunctionoftimeoftheexchangereaction,usingasingleexponentialfit.
Usersmayalsochoosetoexportdatatoenabledeterminationoftherateconstantsofexchangewithotherfitmodels.
ResultsandOutputThebasicoutputofHDXFinderisanhtmltablethatcanbedownloadedasacsvfile.
Thistablelistsallthepeptidesofinterest,orderedaccordingtotheirrelativepositionontheproteinaminoacidsequence,andincludesaminoacidsequence,retentiontime,averagemass,deltamassforeachexperimentaltime/condition,andrateconstantofexchange(ifthatanalysiswasrequested).
Additionally,ifapdbfilewasprovided,HDXFindercanproduceaviewwiththerateconstantsofexchangemappedontotheproteinstructure.
ThisstructurewithpeptidemassinformationcanbevisualizedwithJmolinthewebbrowser.
SeeFigure2usingpyruvatekinaseasanexampleofthisoutput.
DiscussionHDXFinderworksbyfirstextractingallisotopicenvelopesfromanMSexperiment.
Thisprocessisautomaticandisindependentofanyconsiderationforthenatureofisotopicenvelopes(i.
e.
,chemicalcomposition)orfortheirdeuteriumcontent.
Theonlylimitingvariablesconsideredarethattheisotopicpeptideenvelopesshouldhaveameasurablecharge(z=1to6)andachromatographicelutionprofileconsistentwithanelutingion(seeSupplementarymaterial).
Thereareseveraladvantagestoextractingallisotopicenvelopesbeforecorrelatingthemtoindividualpeptides:(1)theextractionofisotopicenvelopesisperformedonlyonce,resultinginasignificantincreaseintheoverallspeedoftheanalyticalprocess.
(2)Theextractionofallisotopicenvelopespresentinagivenexperiment,independentoftheidentificationofthecorrespondingaminoacidsequence,allowsforthereportingoftheisotopicenvelopesofunknownprimarysequence.
Thismayallowtheusertoincreaseproteincoveragebyre-attemptingpeptideidenti-ficationusinganinclusionlistfortheseparticularions.
(3)Theapplicationofthealgorithmsusedforthisprocessresultsintheextractionofoverlappingisotopicenvelopesandtheremovalofspuriousnoise.
Tohelppreventbackexchangeandmaintaindeute-riumlabel,peptidesareelutedquickly(i.
e.
,steepgradientsandhighflowrates)fromHPLCreversed-phasechromatographiccolumns.
However,despitecon-siderableeffortininstrumentandmethoddevelopment[13–16],steepgradientsoftenresultintheco-elutionofpeptideionswithoverlappingisotopicenvelopes[9].
Thismakesitdifficulttodetermineaccuratelytheexperimentalaveragemassofthepeptides.
Asmentionedabove,programssuchHDDesktop[8]andHeXicon[6]determineaveragemassesbyperformingcurve-fittingoftheisotopicenvelopestoatheoreticalenvelope.
IncontrasttootherprogramsthatincorporateanevaluationofHPLCretentiontimesasastepforextractingaveragemassesofpeptides,HDXFinderdoesnotrequire,asaninput,theretentiontimesforthedetectionoftheelutingpeptides.
Instead,allisotopicpeptideenvelopesaredetectedautomaticallyandsub-B-domainA-AinterfaceC-Cinterface0.
001-0.
0400.
041-0.
0600.
061-0.
090>0.
091Figure2.
RateconstantsofH/Dexchangemappedtothemonomerofpyruvatekinasefrommammalbrain/muscle(M1-PYK)(PDBcode2G50).
ThetwotypesofsubunitinterfaceswithinthehomotetramerarelabeledA-AandC-C.
TherateconstantsofH/Dexchangehavebeengroupedinfourcategories:Blue(0.
001–0.
040),green(0.
041–0.
060),orange(0.
061–0.
090),red(90.
091).
Allrateconstantsofexchangearefromsingleexponentialfits—independentlyofthegood-nessofthefit—andareexpressedinmin–1D.
E.
Miller,etal.
:AutomatedAnalysisofH/DExchangeMassSpectrometry427sequentlymatchedandcorrelatedtothepeptidesincludedinthelist.
Theretentiontimealongwiththeintensityoftheion,asmeasuredbyHDXFinder,areutilizedfortherankingofions(seeSupplementarymaterialfordetails).
Themainadvantageofthesetofweb-basedtoolsincludedinHDXFinderistheabilitytoextract,match,correlateandrankisotopicenvelopeswiththepeptidesprovided,atallH/Dreactiontimesinanunattended,automaticmode.
Atthesametime,HDXFindergivestheuseranoptiontore-correlateandre-rankallmatchedpeptidedataasneeded.
Toaidmanualsteps,avarietyofgraphicaltoolsareincluded,whichwillallowtheuserto(Figure3a–d):(1)examineindividualaveragedisotopicenvelopesinFigure3.
Verificationtools.
ToaidintheprocessofverificationseveralgraphictoolshavebeenbuiltintoHDXFinder.
Attheindividualreactionpoint,theusercanview:(a)aplotoftheelutingpeptidecomparedtootherpeptides.
Inthisexampletheviewiszoomedout10%withthepeptideofinterestoutlinedinorange.
(b)Ascatterplotofallindividualscans,indicatingthepositionofisotopicpeaksintheenvelopeunderconsideration.
(c)Theaveragedisotopicenvelope(orange)incomparisonwiththetheoreticalisotopicenvelopeforthepeptideunderconsideration(black).
(d)Athree-dimensionalplotofthescansshowingthepeptideelutingovertime.
Attheoverallrangeoftheexchangereaction[panels(e)and(f)],theusercanplotthechangeinm/zasafunctionoftimeeither(e)asathree-dimensionalplotor(f)averagemasschangeforapeptideionovertimeD.
E.
Miller,etal.
:AutomatedAnalysisofH/DExchangeMassSpectrometry428comparisonwiththetheoreticalenvelopeoftheundeuteratedpeptide;(2)displaytheelutionprofileofanisotopicenvelopeonathree-dimensionalgraph(i.
e.
,m/zversusretentiontimeversusintensity);and(3)makeuseofscatterplotsofm/zversusretentionofisotopicenvelopestoestablishchromatographicelutionprofiles.
Foranalysisacrosstherangeoftheexchangereaction(Figure3e,f),theusercan:(1)displayatwo-dimensionalgraphoftheaveragemassforapeptideionoverthetimeoftheexchangereaction;(2)displaythedeuteriumexchangeprofilesforapeptideionoverthetimeoftheexchangereactionasathree-dimensionalprofileofaveragedisotopicenvelopes(m/zversusabundanceversustimeofexchange).
Insummary,HDXFinderprovidesasimple,fast,andautomatedroutinefortheidentificationofpeptidesinH/DMS.
Withtheseadvantages,thisprogramshouldfindbroadapplicationinthemassspectrometrycommunity.
AcknowledgmentsTheauthorsacknowledgesupportinpartforthisworkbyNIHgrantDK78076awardedtoA.
W.
F.
andbyITSgrantfromtheUniversityofKansastoA.
A.
S.
TheauthorsthankDr.
DavidNualartforvaluablediscussions.
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