differencetube24.com

BAmericanSocietyforMassSpectrometry,2019J.
Am.
Soc.
MassSpectrom.
(2019)30:932Y945DOI:10.
1007/s13361-019-02160-3SubstancePintheGasPhase:ConformationalChangesandDissociationsInducedbyCollisionalActivationinaDriftTubeChristopherR.
Conant,1DanielR.
Fuller,1ZhichaoZhang,1DanielW.
Woodall,1DavidH.
Russell,2DavidE.
Clemmer11DepartmentofChemistry,IndianaUniversity,800KirkwoodAvenue,Bloomington,IN47401,USA2DepartmentofChemistry,TexasA&MUniversity,CollegeStation,TX77843,USAAbstract.
Theworkpresentedbelowisrelatedtoourcompanionpaperinthisissue,entitled:SubstancePinsolution:trans-to-cisconfigura-tionalchangesofpenultimateprolinesinitiatenon-enzymaticpeptidebondcleavages.
Two-dimensionalionmobilityspectrometry(IMS-IMS)andmassspectrometrytechniquesareusedtoinvestigatestructuraltransitionsfor[M+3H]3+ionsofsubstanceP(subP)uponcollisionalactivation(CA)inthegasphase.
Inthisapproach,differentconformationsofionshavingaspecifiedmobilityareselectedafteraninitialIMSseparation,collisionallyactivatedtoproducenewconformers,andtheseproductstructuresareseparatedagainusingasecondIMSregion.
Inthisway,itispossibletofollowfoldingandunfoldingtransitionsofdifferentconformations.
Theanalysisshowsevidenceforfiveconformations.
Unlikeothersystems,everytransitionisirreversible.
Studiesasafunctionofactivationvoltageareusedtodiscernpathwaysofstructuralchangespriortoreachingtheenergyrequiredfordissociation.
Thresholdsassociatedwiththeonsetsoftransitionsarecalibratedtoobtainestimatesoftheenergeticbarriersbetweendifferentstructuresandsemi-quantitativepotentialenergydiagramsarepresented.
Overall,barriersassociatedwithstructuraltransitionsof[subP+3H]3+intheabsenceofsolventareontheorderof~40kJmol1,substantiallylowerthanthe~90kJmol1requiredforsomesimilarstructuraltransitionsinsolutionsofethanol.
Comparisonsofthetransitionenergiesinthegasphasewiththermochemistryforsimilartransitionsinsolutionprovidecluesaboutwhyreversetransitionsareprohibited.
Keywords:Ionmobilityspectrometry-massspectrometry,Peptideconformation,ActivationenergyReceived:22January2019/Revised:15February2019/Accepted:15February2019/PublishedOnline:12April2019IntroductionItisfairtoaskthequestion:whywouldanyonewanttostudytheconformationsofbiomoleculesinavacuumAfterall,thesemoleculesrarelyfindthemselvescompletelystrippedofsolventatpressuresfoundintheupperatmosphere.
And,suchstudiesarenoteasy—requiringcomplexinstrumentationthatoftenneedstobedesignedandconstructedinhouse.
Moreover,today,with~137,000entriesintheproteindatabank,muchisElectronicsupplementarymaterialTheonlineversionofthisarticle(https://doi.
org/10.
1007/s13361-019-02160-3)containssupplementarymaterial,whichisavailabletoauthorizedusers.
Correspondenceto:DavidClemmer;e-mail:clemmer@indiana.
eduFOCUS:IONMOBILITYSPECTROMETRY(IMS):RESEARCHARTICLEknownaboutthenearly1400uniquefoldsthatdescribenativestructures[1,2].
Somuchso,thatamachine-learningapproachdevelopedbytheGooglesubsidiaryDeepMindwonthe2018CASP13protein-foldingcompetition,withthemostaccuratepredictionsof25of43unknownstructures;thenearestcom-petitiveapproachwasmostaccurateforonlythreesequences[3].
Theabilitytopredictnativestructuresfromprimaryse-quencesisamajoradvancethatbuildsonmorethanahalfcenturyofexperimentalmeasurements[4–8].
Onemightimag-inethattheprotein-foldingproblemislargelysolved.
But,nativestructuresareonlyapartofthisproblem.
Proteinssamplemanyothernon-nativeconformationsastheyaresyn-thesized,modified,andtransportedthroughnewenvironments[9].
Littleisknownaboutthesestates.
Non-nativeconforma-tionsmayormaynotfunctioninthesameway,orwiththesameefficiency,asnativestructures[10,11],buttheyarecriticaltolivingsystems.
Inordertomaintainproteostasis,denaturedstructuresmustberecognizedassuch[12],tagged[13],anddestroyed[14],inordertopreventdeleteriousconse-quencessuchasaggregation[15–17].
Wehavepreviouslyquoted[18,19]Lumry'sandEyring'snowclassic1954paper[20],BConformationChangesofProteins^whichbeginsbystating,B[t]hetermproteindenaturationeveninitsoriginalmeaningincludedallthosereactionsdestroyingthesolubilityofnativeproteinsandhassinceacquiredsomanyothermean-ingsastobecomevirtuallyuseless.
^Inthe65yearssince,littlehaschanged.
Inlargepartthisisbecauseitisextremelydifficulttotrap,purify,andcharacterizenon-nativestates.
Oneexceptiontothisdifficultycomesaboutwhensolventisremovedasspeciesaretransferredintomassspectrometers.
EarlystructuralstudiesofnakedbiomoleculesfromFenselau's[21],McLafferty's[22],Douglas'[23],Cooks'[24],Williams'[25],Bowers'[26],andJarrold's[27]groups(amongothers)wereperhapsinitiallydrivenbycuriosity.
But,wemightnowask:whatbetterplaceistheretostudynon-nativestructures,thaninthegasphaseIntheabsenceofalubricatingsolvent,somenon-nativestructuresarestableforlongtimes[28],allowingthemtobeprobedwithanarsenaloffastandpowerfulmassspectrometrictechniquesdevelopedduringthelastcen-tury[29].
Moreover,theevaporativecoolingprocessassociatedwithcreatingmacromolecularionsbyelectrosprayionization(ESI)[30]rapidlyfreezes-outspecificstructuresastheydry[18,31,32].
TheseBfreeze-driedbiomolecules,^asBeauchamp'sgroupcalledthem[33]arenowmorethanacuriosity;theyprovideaccesstonon-nativestateswherefewoptionsexist.
And,studiesofnakedproteinsprovidethechancetoexamineintramolecularinteractionswithoutcompli-cationsduetosolvent[34].
Asmoreinformationbecomesavailable,computationalmethodswillundoubtedlyprovideamoredetailedunderstandingofhowsuchstructuresareformedandwhatfunctionalordysfunctionalrolestheyplay.
Perhapsitisnotallthatsurprisingthatstructuresarestabi-lizeduponremovalofsolvent.
Afterall,removalofsolventishowproteincrystalsarestabilized[35].
And,whilesomeweresuspiciousthatearlycrystalstructuresmaylackkeyaspectsrelevanttosolutionstructure[36],theyappeartohavecaughtonandarenowwidelyaccepted.
Below,wedescribetheuseofESIwithhybridionmobilityspectrometry-massspectrometryandcollisionalactivationtechniques(IMS-CA-IMS-MS)toprobestructuraltransitionsofthesimple,model,andundecapeptidesubstanceP(subP)inthegasphase.
Thispep-tide,awell-studiedmemberofthetachykininfamily[37],hasthesequenceArg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-Phe8-Gly9-Leu10-Met11-NH2.
Recentstudies,usingthecryogenic-IMStechniquespioneeredbyRussell'sgroup[31],foundevidencefortwotypesofconformers:akineticallytrappedstructurethatemergesinthegasphaseuponevaporationofsolvent(con-formerA),andanextendedgasphasestructurethatformsuponannealingdesolvatedsubPions(conformerB).
Inanotherpaperinthisissue,Conantetal.
describekineticsstudiesofstructuralchangesofsubPthatultimatelyresultinnon-enzymaticcleavageofspecificbonds,whensubPisincubatedinethanolsolutions[38].
Inethanol,atrans-Pro2→cis-Pro2configurationalchangeregulatescleavageofthePro2-Lys3peptidebond.
Afterthisoccurs,thesubP(3–11)fragmentthatisformedundergoesasimilartrans-Pro4→cis-Pro4isomeriza-tionbeforethePro4-Gln5bondspontaneouslycleaves.
Inbothdissociationevents,productpeptidesareaccompaniedbyfor-mationofacyclicdiketopiperazine(DKP)dipeptide.
ThisspontaneousprocessingisverydifferentthanenzymaticdipeptidylpeptidaseIVcleavageofpenultimateprolinepeptidebonds[39],whichoccursonlyfromthetrans-configurationandformsdipeptideproductsratherthanDKPs.
Thepresenceofthesesolutionintermediatesandpreservationofkineticallytrapped[subP+3H]3+ionsinthegasphaseprovidesaninter-estingopportunitytoalsocomparestructuralchangesandbondcleavagesinsolutionwiththoseinduceduponcollisionalacti-vationinthegasphase,whichwedobelow.
ExperimentalIMS-CA-IMS-MSMeasurementsTheinstrumentusedforthestudiesdescribedherewasde-signedandconstructedbyKoenigeretal.
andaschematicdiagramisshowninFigure1[40].
Thisinstrumentuseslongdriftregionsandlowpressures.
Eachdriftregionis~300timeslongerthantheexcitationregion,suchthatdifferencesindrifttimesassociatedwiththehigherelectricfieldsintheCAregionaresmallerthanthe60μsbinsizesusedtorecorddrifttimedistributions.
Atlowpressures,collisionalcoolingofactivatedionsoccursmoreslowlythanathighpressures.
Thismakesitpossibletoactivateionsinsideofthedrifttubeusingrelativelylowvoltages.
Ourapproachisverysimilartothenowwidelyusedcollision-inducedunfoldingmethod(CIU,whereionsareinjectedintoadrifttubeatdifferentvoltages)thatwaspioneeredandperfectedbyJarrold's,Bower's,andRuotolo'sgroupsandisnowcommerciallyavailable[41,42].
TheCIUapproachisremarkablysensitivetoverysubtledifferencesinstructuresandstabilities,evenforlargeions[43].
C.
R.
Conantetal.
:SubstancePintheGasPhase:ConformationalChangesandDissociations933ExperimentalIMS-CA-IMS-MSmeasurementsarecarriedoutusingamethodpioneeredbyPiersonetal.
[44]Briefly,ionswereproducedandintroducedtotheIMSdrifttubeusingaTriVersaNanoMate(Advion,Ithica,NY)autosamplerandnanosprayionizationsource.
Thedrifttube[40]consistsofasourceregionthatperiodicallyreleasespacketsofionsfromagriddedelectrostaticgate(G1)intothefirstdriftregion(D1)where,undertheinfluenceofauniformelectricfieldalongtheaxisoftheinstrument,ionsmigratethrougha0.
9-mdrifttubecontaining~3.
0TorrofHebuffergasbeforeenteringacolli-sionalactivationregionwheretheyareactivatedwithanap-pliedvoltage.
Uponexitingthisregion,theions(whichmayhavechangedconformationorundergonefragmentation)enterasecond1.
0-mdriftregionwheretheyundergoasecondseparationpriortodetectioninatime-of-flightmassanalyzer.
Thefirstandseconddriftregionsareseparatedbyanionfunnel(F2)thatservestoradiallyfocusionsandcontainsanelectro-staticgate(G2)thatmayberaisedandloweredperiodicallytoallowionsofaspecificmobilitytopass.
Thefunnelalsocontainsanactivationregion(IA2),operatedforthisexperi-mentatvoltagesrangingfrom6to200V,thatmayrapidlyaccelerateionsforcollisionalactivation.
AfterexitingIA2,ionsarerapidlythermalizedtothebuffergastemperatureandareseparatedagaininD2.
Anionfunnel(F3)thenfocusestheionsbeforetheyexitintothemassspectrometer[45].
DeterminationofExperimentalCollisionCrossSectionsfromIonMobilityDistributionsCollisioncrosssections(Ω)weredeterminedfromiondrifttimes(tD)usingEq.
(1)[46].
Ω18π1=216zekbT1=21mI1mB1=2tDEL760PT273:21N1IncludedinthisequationaretermsforBoltzmann'sconstant(kb),temperature(T),chargeoftheion(z),elementarycharge(e),massesoftheion(mI)andbuffergas(mB),andtheneutralnumberdensityofthebuffergasatstandardtemperatureandpressure(N).
Theelectricfield(E),thelengthofthedrifttube(L),andpressure(P)aredefinedexperimentally.
TheIMS-CA-IMS-MSinstrument(Figure1)isde-signedsothatcrosssectionscanbemeasuredinseveralways.
Themostaccuratemeasurementisobtainedbyscan-ningthedelaytimeassociatedwithreleaseofionsfromtheG1andselectionofionsatG2acrossapeak.
Inthisregion,theelectricfieldisuniform,thelengthofthedriftregionisnearlyexactlydefinedasthedifferencebetweenthegridsofG1andG2,andthedrifttimeavoidsinclusionofanytimethationsspendoutsideofthedriftregion(e.
g.
,timeassociatedwithtransferofionsintothesourceoftheMS)aswellasflighttimesofionsintheMS.
Thisapproachcanbeusedtocheckmobilitiesthattravelthroughtheentireinstrumentandcreateacalibrationcurveforcross-sectionaldistributionsrecordedusingtheentireD1andD2regions.
Finally,thedrifttimecanbemeasuredwithrespecttotheselectiongateG2,allowingcrosssec-tionstobedeterminedforactivatedions.
PeptideSynthesisandSamplePreparationSubstancePwasobtainedfromSigmaAldrich(≥95%purity,St.
Louis,MO).
SeveralsubPanaloguesinvolvingarangeofPro→AlasubstitutionsweresynthesizedusingstandardFMOCsolid-phasepeptidesynthesiscar-riedoutonanAppliedBiosystems433APeptideSyn-thesizer(AppliedBiosystems,FosterCity,CA)[47].
Peptidesolutions(10μMin1-propanol)wereelectrosprayedusingaTriVersaNanoMateautosampler.
Wefocusedthesestudiesonionsproducedfrom1-propanolbecausethissolventproducesfourstructuresthatappeartobetrappedduringtheelectrosprayprocess.
Thus,thissystemallowsustostudytransitionsofdif-ferentstructuresofthesamepeptide.
Figure1.
DiagramoftheIMS-IMS-MSinstrumentemployedinthesestudies934C.
R.
Conantetal.
:SubstancePintheGasPhase:ConformationalChangesandDissociationsMethodforInvestigatingcis/trans-ConfigurationsofPro2andPro4PeptideBondsProlineisuniqueamongthenaturallyoccurringaminoacidsbecausethepyrrolidinesidechainrestrictsforma-tionofthemorecommonlyadoptedandenergeticallyfavorabletrans-configuredpeptidebond[48–52].
Asaresult,prolinehasanincreasedtendencytooccupythecis-form,whichfrequentlyleadstoadditionalstructuralfeatures[53–55].
Substitutionofanalanineresidueforaprolineresiduepreventsformationofacis-configuredpeptidebondthatcanbeformedbyproline.
Thus,com-parisonsofthecross-sectionaldistributionsforsubP(containingproline)withdistributionsrecordedforPro→Ala-substitutedanaloguesallowustoobtaininsightabouttheconfigurationoftheprolinepeptidebondcon-figuration.
Tomakethiscomparison,itisusefultoaccountfordifferencesinthecrosssectionsthatarisefromdifferencesinthesizesofprolineandalanineresidues.
Thisdifferenceisknownfromvaluesofintrin-sicsizeparameterswhichwereinitiallydeterminedforallaminoacidsbyCountermanandValentine[56–60].
Withthesecorrections,Piersonetal.
assignedthecis-andtrans-configurationsofeachprolineresidueintheconformationsofthebradykininpeptidebackbonecon-figuration[61].
Similarly,Fortetal.
utilizedalanine-substitutionofvarioussubPresidues(Pro,Gln,Phe)tocharacterizethekeyresiduesinstabilizingconformerAof[subP+3H]3+[62].
Below,weanalyzethreePro→Alasubstitutedsequences:RAKPQQFFGLM-NH2[subP(P2A)],RPKAQQFFGLM-NH2[subP(P4A)],andRAKAQQFFGLM-NH2[subP(P2,4A)].
StudiesofAla-substitutedanaloguesasafunctionofactivationenergyallowustoidentifytheoriginofspecificstructuralchanges.
CalibrationofThresholdVoltagesToObtainActi-vationEnergiesActivationvoltagesarecalibratedtoobtainactivationenergies,asdescribedpreviously[44].
Briefly,collisionalfragmentationthresholdvoltagesfrommeasurementsinthedrifttubearecalibratedtoreportedthermochemistry.
Mostofthethermochemistryusedtocalibrateourmeth-odwasdeterminedbyArmentroutwhohaspioneeredthemostrigorousstatisticalanalysesassociatedwithdeter-miningfragmentationthresholdsfromsingle-collisioneventsthatleadtonewions(eitherfragmentsorprod-uctsofion-moleculereactions)[63–65].
ThecalibrationalsousesthermochemistryforbradykininionsfromanArrheniusanalysisofdissociationratesmeasuredinatemperature-controllediontrapbyMcLuckeyandhiscoworkers[66],andanaverageofseveralreportsofthermochemistryforleucineenkephalin[67–70].
Eachexperimentalthreshold,definedasthevoltageatwhichaproductstateabundancereaches1%normalizedabun-dance,ismultipliedbycharge,dividedbynumberofvibrationaldegreesoffreedom(d.
o.
f.
)oftheactivatedspecies,andcalibratedtoliteraturevaluesforthedisso-ciationenergiesasshowninEq.
(2)[44].
Ea1:590Vzd:o:f:0:0392ThecalibratedthresholdsdeterminedfromEq.
(2)areinunitsofeVandthesevaluesarereportedinkJmol1.
Theuseof1%relativeintensityasthresholdvoltageswasnotbasedonstatisticaltheory,butwaschosenasthepointofasignal-to-noiseratiosufficientforconfidentdetection,describedprevi-ously[44].
Otherdefinitionsofthethreshold(e.
g.
,2%or5%)couldbecalibratedandusedfordeterminationofthresholds.
Oncecalibrated,otherdefinitionsyieldsimilarvalues[71].
Onecriticalcaveatofthisapproachisthatitdoesnotcapturetheeffectsofentropy.
Specifically,itassumesthattransitionstateshavesimilarentropiesofactivationwhenapproachedfromtheforwardorreversedirections.
Thatis,theyarebotheithersimilarlylooseortight.
Thisassumptionappearstobevalidforactivationofthemainpeaksobservedinthequasi-equilibriumdistributionforbradykinin.
But,thisisclearlynotthecaseforsubstanceP.
Thestudiesdescribedbelowrevealthatfourconformersoriginatefromsolutionandifprovidedenoughactivationenergy,eachofthesewillformtheconform-erB—thegas-phasestructure.
But,noneoftheseprocessesisreversible.
Thisstronglysuggeststhatsolventisrequiredtoreachthetransitionstatesnecessarytoformtheseconformers.
Thisfindingintroducesanimportantcaveat.
Below,wereportthresholdenergiesandtreatthemastransitionstateenergiesfortheforwarddirectionthatresultsinformationofB;however,strictlyspeaking,thesevaluesareupperlimitstothetransitionstateenergiesandmayalsobesubjecttokineticshiftslargerthanthereactionswithloosetransitionstatesthatwereusedtocalibratethismethod,andsothesereactionsmaygiveanactivationenergythatisslightlytoolarge.
Interestingly,ourreportedvaluesforthesetransitionsarerelativelylow,~28to54kJmol1,especiallywhencomparedwiththesolutionthermochemistryforsimilartransitions.
Therefore,itseemslikelythatkineticshiftsintheforwarddirectionaresmall.
ResultsandDiscussionIMSCross-SectionalDistributionsfor[subP+3H]3+Figure2showsatypicalcross-sectionaldistributionfor[subP+3H]3+ionsobtaineduponelectrosprayingfromasolu-tionofpropanol.
ThemostabundantpeakinthespectrumatΩ=3002correspondstoaconformationthatwasobservedpreviouslybyRussellandcoworkersandiscalledconformerA[31].
Oneofthesmallerpeaks,centeredatΩ=3542wasalsoobservedpreviouslyandiscalledconformerB[31].
Addition-ally,wefindevidencefortwonewverylow-intensitypeakscenteredatΩ=3332and3392,whichwehavelabeledasC.
R.
Conantetal.
:SubstancePintheGasPhase:ConformationalChangesandDissociations935C1andC2,respectively,thatwediscussinmoredetailbelow.
Theobservationofmultipleconformationsforthispeptideissimilartoresultsforotherpeptideswithprolineresidues,whichoftenshowevidenceformultiplestructuresassociatedwithprolineresiduessamplingbothcis-andtrans-peptide-bondconfigurations[53–55].
SelectionandActivationofIndividualConformersToexplorestructuralchangesinthissystem,eachpeakwasselectedbasedonitsmobilityinthefirstdrifttubeandsubject-edtoCAatdifferentactivationvoltages.
Theresultsforselec-tionandactivationforeachconformerareshowninFigure3.
Webeginbydiscussingthemostabundantspecies,conformerA.
Uponselectionandactivation,thispeakremainstheonlyfeatureinthedistributionbelow~50V.
Atanappliedactiva-tionvoltageof56V,weobserveinFigure3thatasmallfractionoftheconformerAionsunfolds,formingconformerB,whichhasalargercrosssection.
AstheCAvoltageisincreased,thedistributionshiftstofavorBandby68V,Bdominatesthedistribution,becomingtheonlyobservablefea-tureaboveCA~70V.
ThisresultisconsistentwithresultsreportedbyRussell'sgroup,whereionswereactivatedinthesourceregion[31].
Previously,wereportedthatathighactivationvoltages(priortofragmentation),bradykininionsfavoraBquasi-equi-libriumdistribution^(QED)[72].
Thatis,whentheactivationenergyexceedsallofthebarriersbetweendifferentstructures,increasingactivationvoltagenolongerresultsinchangestothepopulationsofdifferentstatesthatarepresent[44,72].
Addi-tionally,theQEDdistributionofbradykinin(whichinvolvedthreemainstructures)canbereacheduponactivatinganyofthesixresolvedstructuresthatwereproduceddirectlybyESIforthision.
InthecaseofsubP,onlyasinglepeakisobservedathighenergies.
Thispeak(conformerB)maybecomprisedofmultiplestructureswithsimilarcrosssectionsthatarenotresolvedandreflecttheQEDofgas-phasesubPionsthatappearstohavebeenreachedat~70V.
ConformerAisnotobservedintheQED.
WeinterpretthisasanindicationthatconformerAresultsfromapopulationofstatesthatarekinet-icallytrappedasionsemergefromsolution.
Thisisconsistentwiththecryogenic-IMSmeasurements[31].
Analogousselectionandactivationexperimentswerecar-riedoutforthesmallerpeaks(B,C1,andC2)asshowninFigure3.
WhileconformerBdominatesthedistributionwhenformedathighactivationenergiesfromconformerA,onlyasmallpopulationisformeddirectlyfromthesource.
IntegrationoftheionsignalsinFigure2indicatesthatconformerBcomprisesonly~1.
5%ofthetotaldistribution.
SelectionandactivationoftheΩ=3542conformerBpeakresultsinaninterestingsetofdistributions.
Mostoftheseions(>98%)donotappeartochangestructureuponactivation.
Thisisconsis-tentwiththeideathatinthegas-phaseBionsaremorestablethanAions.
Inthiscase,wesuspectthattheconformerBionsobserveddirectlyfromoursourceareformedbyactivationofaconformerA,aftertheiremergenceintothegasphaseasions(presumablythisslightactivationoccursintheionfunnelregionofoursource,inanalogytoRussell'sactivationresults)[31].
ActivatingtheΩ=3542peakat60VshowsevidenceforaverysmallpopulationofconformerAions.
Itappearsthat~2%oftheselectedΩ=3542ions(whichinitiallycom-prisedonly~1.
5%ofthedistributionofionsfromthesource)canformconformerA.
However,astheactivationenergyisincreasedbeyond~80V,thispopulationvanishesandonlyBisobserved.
Thisresultrequiresthatanadditional,verylowabundanceconformermustbepresent.
UnlikeotherBions,uponactivation,thissmallpopulationofspecies(whichwecallconformerB*)mustbekineticallytrapped(similarlytoA)anduponactivationtheseionsarecapableofformingconformerA.
TheAstatethatisproducedfromB*presumablyexistsoveranarrowrangeofenergies(asshownbelow)becauseathigherenergies,AcanconverttoB.
TheB*conformertrappedduringtheESIprocessrepresentsonly~0.
03%(2%*1.
5%)oftheinitialdistributionofions.
ThisanalysisnotonlyillustratesthevalueofselectingandactivatingionsbyIMS-IMSasameansofrevealingdifferencesinstructuresthathaveidenticalcrosssectionsbutalsohighlightsthehighsensitivityofthesemethods.
Figure2.
Mobility-separated,cross-sectionaldistributionfor[subP+3H]3+measureduponelectrosprayingsubPfrometha-nol.
ThemajorpeakcorrespondstoconformerA,asassignedpreviously[31].
Threeverylowabundancepeaksarealsoob-served,correspondingtoconformersC1,C2,andB.
Theregionassociatedwithlowabundancestructuresismultipliedbyafactorof20(dashedline)936C.
R.
Conantetal.
:SubstancePintheGasPhase:ConformationalChangesandDissociationsFigure3alsoshowscross-sectionaldistributionsthatarerecordeduponselectionandactivationofC1andC2ions,whicheachcomprise~0.
5%oftheinitialsourcedistribution.
BothoftheseionsformconformerAatintermediatevoltages.
AsobservedforB*,athighervoltagesAdisappearsandonlythefinalBproductremains.
TheformationofAfromC1andC2suggeststhatthesearealsokineticallytrappedstructuresfromsolution.
Nootherstructuresformeitherofthesespeciesinthegasphase,consistentwiththisidea.
CarefulexaminationofFigure3revealsaverysmalldiffer-encebetweentheC1andC2conformers.
Atintermediatevoltages,e.
g.
,38VinFigure3,theC2peakshowsasmallshoulderataslightlylargercrosssections(Ω=3542)con-sistentwithformationofBorB*.
Asthecollisionvoltageisincreasedthisshoulderdecreasesinabundance,disappearingentirelyby~48V.
ThisbehaviorisconsistentwithformationofB*.
AthigherCAvoltages,thepeakatΩ=3542returnsanddominatesthedistribution.
ThisistheBconformer,anditbecomestheonlyproductobservedabove~70V.
ItisimportanttonotethatRussell'scryogenic-IMSresultsshowdefinitivelythatconformerAemergesdirectlyfromsolution,uponevaporationofthelastremainingsolventmole-culesfromtheion.
But,here,wehaveshownthatselectionandactivationofC1,C2,andalsoB*inthegasphasecanformastatewiththesamecrosssectionasconformerA.
TheonlystructurethatdoesnotformAuponactivationisconformerB.
ThisrequiresthateitherconformerAcanbeformedinthegasphasefromactivationofotherstructuresorthatgas-phaseactivationofotherconformersproducesadifferentspecieswiththesamecrosssectionastheconformationthatemergesdirect-lyfromsolution.
ThesameistrueofB*.
ActivationofC2showsthatB*canbeformedthegasphase.
However,theobservationthatB*formsAindicatesthatB*isakineticallytrappedconformerandcouldalsoemergedirectlyfromsolution.
ChangesinConformerAbundancesasaFunctionofActivationVoltageAsummaryoftheabundancesthatareobtainedfordifferentstructuresuponselectionandactivationofeachofthecon-formersatalloftheCAvoltagesusedinthesestudiesisshowninFigure4.
Thesedataareconsistentwiththechangesinthepeaksdiscussedabove.
Figure4alsoshowssimplereactionmechanismsthatareconsistentwiththediscussiongivenaboveuponactivatingeachion.
Oneimportantfindingisthatthetransitionsdescribedaboveshownoevidenceofbeingrevers-ible.
AscanbeobservedfromFigure4,asthecollisionvoltageisincreased,from~50to80V,thepopulationofconformerAionsdecreasesasBincreasesandnofurtherchangesareobserveduntilfragmentationisobservedat~90V.
WhentheΩ=3542peak(dominatedbyB)isselectedandactivated,weobservethesmallpopulationofB*thatformsA.
Athigherenergies,AformsB.
Again,thelargepopulationofBionsdoesnotchangeoverawidedistributionofenergies(herefrom0to90V)untilthethresholdforfragmentationisreached.
Similarly,thelowestenergyproductobserveduponactivationofC1isAandathigherenergiesBisobservedFigure3.
IMS-CA-IMScross-sectionaldistributionsformobility-selected[subP+3H]3+conformersuponactivationintheIA2region(seetextfordetails).
Eachofthefourconformers(A,B,C1,C2)showninFigure2wasselectedandcollisionallyactivatedusingthevoltagesthatareindicatedinthefigureC.
R.
Conantetal.
:SubstancePintheGasPhase:ConformationalChangesandDissociations937priortofragmentation.
ActivationofC2issomewhatdiffer-ent.
ThisconformerformsB*andA.
AthigherenergiesB*alsoformsAandatevenhigherenergiesconformerBdominatesbeforedissociation.
ExtractionofThresholdsandDeterminationofActivationEnergiesforStructuralTransitionsFigure4alsoshowsadetailedplotofC2activationshowingthethresholdregionsforeachtransition.
Similaranalysisof1%thresholdsforeachactivatedconformeryieldactivationener-giesforeachtransition.
ThesevaluesaresummarizedinTable1,andasimplerepresentationofthereactioncoordinateassociatedwiththeseconformationalchangesisshowninScheme1.
Thisanalysisrevealsthatbarriersforthesegas-phasetransitionsareintherangeof~28to54kJmol1.
FragmentationThresholdsandDissociationEnergiesFigure4alsoshowsthatatveryhighenergies(above~90V),theBconformersfragment.
Thefragmentationproductsthatareobservedareidenticalregardlessofwhichconformerisselectedforactivation.
Thisisnotsurprising.
AsionsentertheactivationregionIA2theyundergoarapidheatingandcoolingprocess.
Thisisarelativelyslowcycle(ascomparedtoisomerization);weanticipatethateveryconformerwillconverttoBpriortodissociation.
Figure4.
(Left)Relativeabundanceplotsof[subP+3H]3+conformersA,B(andB*),C1,andC2.
Observedtransitionpathwaysareshownforeachconformer.
Amplifiedabundancesoflow-intensityions(i.
e.
,B*formedbyC2andAformedbyB*)areincludedforclarity.
(Right)Expansionofthe0–5%abundancerangefromactivationoftheC1ions,showingabundancesassociatedwithformationofeachproduct(A,B*,andfragments).
Thedashedlinesindicatethe1%abundancethresholdsforeachtransitionandtheapproximatethresholdvoltage938C.
R.
Conantetal.
:SubstancePintheGasPhase:ConformationalChangesandDissociationsFigure5showsfragmentationmassspectraatseveralener-gies.
Thelowestenergyprocessinvolveslossofammoniatoformasmallpopulationof[subP-NH3+3H]3+ions.
Whilewecannotunambiguouslyassignwheretheseproductsareformed(thereareseveralamidegroupsassociatedwiththispeptide),lossofammoniainvolvesaveryspecifictransitionstate.
Re-gardlessofitsorigin,thisprocessisentropicallydisfavored,consistentwiththerelativeinefficiency,observedexperimen-tallyasasmallpeakinFigure5.
Athigherenergies,directbondcleavageleadstoformationoftheb102+ion.
Assoonasthisbecomesenergeticallyaccessible,thisprocessdominatesthemassspectrum.
Astheactivationvoltageisincreasedbeyond~130V,weobserveathirdfragment,correspondingtotheb92+.
Thisfragmentcompetesdirectlywithb102+indicatingthatb92+isformedfromb102+inasequentialprocess.
Calibratedthresholdsareusedtoobtaindissociationener-giesassociatedwithtwofragmentationpathways:process1[subP-NH3+3H]3++NH3,whichlikelyrequiresasignificantintramolecularrearrangementinformationofaveryspecifictransitionstatethateliminatesammoniawithoutcleavinganypeptidebond,andprocess2,inwhichapeptidebondiscleavedtoproducetheb102++y1+productions.
AmechanismforNNH2OR1OsubstancePLys3Pro4Pro2-Lys3peptidebondcleavageNNHOR1O+NNH2OR3OsubP(3-11)(cis)(cis)NNHOR1O+NNHOR3OsubP(5-11)H2N+Pro4-Gln5peptidebondcleavageGln5Gln6Phe7Phe8Gly9Leu10Met11NH2cRPcRPcKPGln5Gln6Phe7Phe8Gly9Leu10Met11NH2Gln5Gln6Phe7Phe8Gly9Leu10Met11NH2Scheme1.
SimplereactioncoordinateshowingtheconformationalchangesinsubstancePsampledbycollisionalactivationTable1.
ThresholdVoltagesandCalculatedActivationEnergiesfor[subP+3H]3+TransitionsConformerSelectedaFormedbVoltagecThresholdEad,kJmol1AB48±142±1C1A32±229±2B48±242±2C2B*32±129±1A31±628±5B43±438±4B*A51±444±3B63±254±2A[subP-NH3+3H]3+85±271±2C1[subP-NH3+3H]3+92±576±4C2[subP-NH3+3H]3+90±475±4B[subP-NH3+3H]3+96±579±4A[b10+2H]2+92±476±4C1[b10+2H]2+102±685±5C2[b10+2H]2+105±187±2B[b10+2H]2+105±587±4aMobilitypeakof[subP+3H]3+selectedforactivationbStructureformedduringactivationoftheselectedpeakcCollisionalactivationvoltageappliedintheactivationregionIA2.
TheindicateduncertaintiesinvoltagerepresentthestandarddeviationfromtriplicatemeasurementsdActivationenergythresholdoftheindicatedtransition,calibratedwithEq.
(2).
Activationenergyuncertaintywasdeterminedthroughpropagationofvoltageerrorandtheuncertaintyoftheenergycalibrationfromref[44]C.
R.
Conantetal.
:SubstancePintheGasPhase:ConformationalChangesandDissociations939process2hasbeenproposedinwhichthebackbonecarbonylcarbonofLeu10issubjectedtonucleophilicattackfromthecarbonyloxygenofGly9,arelativelylocalizedrearrangementleadingtopeptidebondcleavageandanoxazoloneintermedi-atefragmentthatrearrangestothefinalb102+product[73].
Wedonotanalyzethethresholdsassociatedwithadditionalprod-uctsformedathigherenergiesfromfragmentationofb102+(e.
g.
,theb92+,andothersmallerfragments,notshown)becausetheseappeartobeformedinsequentialprocesses.
Thebondenergiesthatweobtainfromthisanalysisareinteresting.
Whiletheproductsofdissociationareindepen-dentoftheinitialselectedconformerthatisactivated(indi-catingthatfragmentsareformedafterformationofthedistributionofBions),theenergyrequiredfordissociationdiffers.
Thatis,thisanalysisissensitivetosubtledifferencesinstabilitiesofdifferentprecursorstructures.
Theactivationenergiesnecessaryfordissociationviapathways1and2measuredfromselectionandactivationofeachprecursorconformeraretabulatedinTable1.
Theenergyrequiredtoeliminateammoniaviaprocess1rangesfromalowestvalueof71±2kJmol1forconformerAtoahighestvalueof79±4kJmol1forconformerB.
Itisinterestingtonotethatthesevaluesaresimilartothevalueof76±3kJmol1measuredbyMcLuckeyandcoworkersforeliminationofwaterfrombradykinin,aprocessthatweexpecttobeenergeticallysimilartoeliminationofammonia.
Table1alsolistsvaluesassociatedwithprocess2,whichresultsinformationofb102+.
Theenergeticsassociatedwiththisfragmentationarealsodependentuponwhichprecursorionhasbeenactivated.
Ourthresholdanalysisyieldsdissociationenergiesof76±4kJmol1forconformerA,85±5kJmol1forconformerC1,87±2kJmol1forconformerC2,and87±4kJmol1forconformerBarelistedinTable1.
Comparisonofthesevalueswiththosereportedaboveforprocess1revealsthattheformerprocess(eliminationofammonia)isenergeticallyfavorableby~5to12kJmol1.
Interestingly,whileeliminationofammoniaisfavoredenergetically,assoonasfragmentationpathway2isaccessible,formationofb102+dominatestheproductdistribution,suggestingthatprocess2ismorefavoredentropically.
AssignmentofProlineConfigurationsforDifferentConformersAsmentionedabove,substitutionsofAlaresiduesforProresiduesallowedPiersonetal.
toassignthecis/trans-configu-rationsofdifferentbradykininconformers.
WehavecarriedoutanalogoussubstitutionsofthePro2andPro4peptidebondsfordifferentconformationsofsubP(cross-sectionaldistributionsshowninFigure6)andfindthatassignmentsbasedoncom-parisonsofcrosssectionsalonearesomewhatambiguous.
Still,itisinstructivetogothroughthisanalysisassomeinsightisgainedbyanalyzingthesubP(P2A),subP(P4A),andsubP(P2,4A)Ala-analogues.
WebeginbyconsideringtheprolineconfigurationsforC1andC2,becausetheseassignmentsarerelativelystraightforward.
NoneoftheAla-analoguesformtheC1orC2conformers,withinFigure5.
(Left)Massspectracorrespondingto,frombottomtotop,theselectionofconformerBfrom[subP+3H]3+ions,andresultingfragmentionsfromCAat105V,135V,and156V.
(Right)NormalizedabundancesofconformerBandproductionsasafunctionofvoltage940C.
R.
Conantetal.
:SubstancePintheGasPhase:ConformationalChangesandDissociationsourdetectionlimits(S/N~104intheseexperiments).
Thisindi-catesthateachoftheseconformershasacis-Pro2andcis-Pro4configurationandthetrans-peptidebondsassociatedwiththePro→Alasubstitutionsprohibittheirformation.
Cross-sectionalmeasurementsfortheAla-analoguesofcon-formerA(whencorrectedforthedifferencesinsizeoftheAlaandProresidues,~2.
52)[60]yieldvaluesofΩ[subP(P2A)+3H]3+=3052,Ω[subP(P4A)+3H]3+=3002,andΩ[subP(P2,4A)+3H]3+=2992,oranaveragesize-correctedvalueofΩ(A)=301±22(Figure6).
Atrans-Pro2andtrans-Pro4configurationalassignmentforconformerAisconsistentwiththeideathatcrosssectionsfortheAla-analoguesandΩ(A)=302±22for[subP+3H]3+withnoAlasubstitutionsareidenticalwithintheexperimentaluncertainties(whenconform-erAisproducedfroma1-propanolsolution).
Itisimportanttonotethatwhenelectrosprayedfromethanol[38],thepeakassoci-atedwithconformerAbecomesbroader.
Itislikelythatthisbroadeningisassociatedwithapopulationofionshavingacis-Pro2configuration,asthisisrequiredforDKPformationinsolution[74].
Thus,thetrans-Pro2andtrans-Pro4configurationalassignmentofpeakAfrom1-propanolbasedoncrosssectionsaloneisnotverysatisfying.
ThisassignmentisstrengtheneduponexaminingtheCAdata(seesupportinginformation).
Whenactivated,eachofthethreeAla-analoguesconvertentirelyintoconformerB;more-over,thethresholdsforeachofthesetransitionsare~6kJmol1lowerthanforactivationof[subP+3H]3+withnosubstitution.
SubstitutionoftheAlaresidueimposesatrans-configuredpeptidebondbecauseitraisesthebarrierforformingthecis-configuration[48,49].
Thus,thelowerthresholdsobservedfortheAla-substitutedpeptidescorroboratethetrans-Pro2andtrans-Pro4configurationalassignmentofA.
ConformerBisproducedinthegasphase.
ForsomeoftheAla-analogues,wemustactivateAinordertoproduceB.
AnaverageofthesizeparametercorrectedcrosssectionforallthreeAla-analoguesisΩ(B)=349±12,avaluewhichis1.
7%smallerthanΩ(B)=354±32measuredforsubPwithnoAlasubstitutions(Figure6).
Thisisslightlyoutsideofthe±1%relativeuncertaintythatweexpectforidenticalstructures,suchthatassignmentbasedoncomparisonsofcrosssectionsisalittleambiguous.
ThesimilarvaluessuggestthatconformerBhasatrans-Pro2andtrans-Pro4configuration.
WenotethattheAlaandProsizeparametersvarywithpeptidesize(aswellaspeptidestructures)[59]andothersizeparametervalueswouldyieldslightlydifferentcorrectedcrosssectionsfortheAla-analogues.
Moreover,thisanalysisassumesthattheonlychangeinsizearisesfromthedifferencesintheseresidues,andclearlythissubstitutioncouldaltertheoverallstructureofthisconformerwithinthisrange.
Asmentionedabove,thelowerthresholdsforformingBfromAfortheAla-substitutedpeptidesindicatesthatB(forsubP)hasatrans-Pro2andtrans-Pro4configuration.
Finally,activationoftheΩ(B)=3492peakforeachoftheAla-analoguesprovidesameansofassigningtheprolinecon-figurationsforB*.
TheB*conformerisobservedonlyforthesubP(P2A)analogue,withanenergydependenceandpopula-tionthatisverysimilartosubPhavingnoAlasubstitutions.
Thus,weassigntheprolineconfigurationsoftheB*subPconformerastrans-Pro2andcis-Pro4configurations.
Table2providesasummaryoftheprolineconfigurationsforeachofthesubPconformations.
Table2.
ProlinePeptideBondConfigurationsinEachConformerof[subP+3H]3+[subP+3H]3+conformeraPro2Pro4AtranstransC1ciscisC2ciscisB*transcisBtranstransaConformationsA,C1,C2,B*,andBcorrespondtopopulationsofionsfromtheionmobilitydistributionof[subP+3H]3+ionselectrosprayedfromasolutionof1-propanolFigure6.
Cross-sectionaldistributionsof[M+3H]3+corre-spondingtosubPandPro→Ala-substitutedanalogues.
Thecrosssectionsoftheanalogueswereshiftedaccordingtovaluesofintrinsicsizeparameters,asdescribedintheexperi-mentalsectionC.
R.
Conantetal.
:SubstancePintheGasPhase:ConformationalChangesandDissociations941Semi-quantitativePotentialEnergyDiagramfor[subP+3H]3+ConformersTheactivationenergiesinTable1fortransitionsbetweenstruc-turesandfragmentationofeachselectedstructure(withtheex-ceptionofB*)canbeusedtoconstructthesemi-quantitativepotentialenergydiagramshowninFigure7.
Theenergylevelforeachconformer(exceptforB*)couldbepositionedbyusingthefragmentationpatternsorthresholdsforstructuraltransitionsbetweenstates.
Thus,thereareseveralwaystoproducethisdiagram.
Weshowonlyone,whicharisesasfollows.
InFigure7,wedefinetheenergyofeachstatewithrespecttoconformerB,Figure7.
Energydiagramof[subP+3H]3+derivedfromexperimentalthresholdEabarriersforstructuraltransitionsandthelowenergyfragmentationproducts,[subP-NH3+3H]3+andb102+ions.
Irreversibletransitionsareshownbyablackarrow.
Aproposedtransitionpathwayisshownatthetopofthediagram,whichincludesexperimentallydeterminedcis-andtrans-configurationalassignmentsofeachProresidueforeachstructure942C.
R.
Conantetal.
:SubstancePintheGasPhase:ConformationalChangesandDissociationsbecauseitismoststable.
ThedifferencebetweenfragmentationthresholdsforAandB(Table1)placesAat8±5kJmol1.
ThethresholdfortheA→Btransitionis42±1kJmol1,or50±5kJmol1higherthanB.
Similarly,fromtheexperimentalthresholdsrequiredtoformconformerB,wedeterminetherelativeenergiesofC1,C2,andB*tobe8±5kJmol1,12±6kJmol1,and3±5kJmol1,respectively.
ThebarriersassociatedwiththeremainingtransitionsaretakenfromthethresholdsforeachtransitionthataregiveninTable1:38±6kJmol1fortheC1→Atransition,41±8kJmol1forC2→A,and41±6kJmol1forC2→B*aswellasB*→A.
ThethresholdforfragmentingconformerB,79±4kJmol1,isalsoshown.
AcrosscheckoftheenergiesassociatedwiththeC1andC2conformerscanbemadebycomparingthefrag-mentationthresholdsthatarepredictedfromthisenergydiagram(thatarereferencedtothebarrierformakingB)tothethresholdsthatwemeasureexperimentally.
Thediagrampredictsfragmentationthresholdsof71±6kJmol1and67±7kJmol1forC1andC2,respec-tively.
Experimentally,wefindafragmentationthresholdof76±4kJmol1forC1,inagreementwiththevaluescalculatedfrombarriersforstructuraltransitions.
TheexperimentalthresholdforfragmentationofC2is75±4kJmol1.
WhilethethresholdforC2calculatedfromFigure7is8kJmol1lowerthanmeasuredexperimen-tally,wenotethatwithinthecombineduncertainties,theyareinagreement.
Thisagreementprovidesacrosscheckofthebarrierheightsforstructuraltransitions.
IftherewasasignificantkineticshiftassociatedwithformingB,thenthecalculatedfragmentationthresholdforC2wouldbesignificantlyhigherthanwasmeasuredexperimentally.
ComparisonsofStructuresandStabilities,Struc-turalTransitions,andFragmentsforGas-Phase,Solution-Phase,andEnzyme-BoundsubPThedatapresentedaboveprovideanopportunitytocomparestructuraltransitionsacrossarangeofenviron-ments.
Proline-containingpeptideshavebeenstudiedex-tensivelybecausecis/trans-isomersintroduceasignificantstructuralheterogeneity[53–55].
Anumberofendo-andexo-peptidasesareprolinespecific[75].
Dipeptidylpep-tidaseIV(DPPIV)isespeciallyrelevanttotheworkpresentedhereasittargetspeptidescontainingtrans-configuredpenultimateprolinemotifsandcatalyzestheeliminationXxx-Prodipeptides[39].
Withoutenzymes,insolution(ethanol),onlythecis-configuredPro2caneliminateDKP[38,74].
Therate-limitingtrans→cisisomerizationforsubPreportedbyConantetal.
hasafree-energybarrierof88±6kJmol1.
Basedontheirfindings,ConantspeculatedthatonebiologicalroleofDPPIVmaybetofavorformationofdipeptidesratherthanDKPproducts,whichavoidsthebioactivityofDKP[76].
Inthegasphase,thePro2trans-configurationoftheBstateofsubPisenergeticallyfavored.
Thisconfig-urationcannoteliminateDKP,andunlikeenzymaticprocessingtoformdipeptides,uponactivation,weob-serveeliminationofammoniaandfragmentationattheC-terminalendofthepeptidetoproducetheb102+.
Thus,activationofthegas-phaseionsleadstofragmentsthatarenotobservedinsolutionoruponenzymaticdigestion.
And,thesolutionandenzymaticfragmentsarenotac-cessibleinthegasphase.
Onefinalnoteinvolvestheirreversiblenatureofthetrans-Pro→cis-Protransitionsinthegasphase.
Ourresultsindicatethatalargeentropicbarrierprohibitsthisprocess.
And,weknowthatfromoursolutionstudiesthattrans-Pro→cis-ProtransitionsareobservedpriortoDKPformation.
Thisimpliesthatadditionofethanolincreasestheaccessibilityofthetrans-Pro2→cis-Pro2transitionstate.
Whilethismightbeso,wenotethatinethanol,ConantreportsavalueofΔH=41±5kJmol1andΔS=157±12Jmol1K1forthistransition.
Thatis,thistransitionisextremelydifficulttoreachinsolu-tionaswell.
Itisperhapsnosurprisethatinmostbiologicalsystems,thetrans-configurationofprolineisheavilyfavored.
ConclusionsIMS-CA-IMS-MStechniqueswereusedtocharacterizefiveconformationsof[subP+3H]3+.
Thereisevidencethatallfivestructuresareproducedduringtheelectrospraydroplet-dryingprocess.
Oneisthelowestenergygas-phasestructureB,andtheotherfourarekineticallytrappedconformationsthatcanbeconvertedtoBbycollisionalactivationinthegasphase.
Eachtransitionofthekineticallytrappedstructureswasfoundtobeirreversible.
Thisindicatesthatsolventisrequiredtoapproachkeytransitionstatesinreverse.
Asemi-quantitativepotentialenergydiagramisderivedfromthresholdactivationvoltagesthatarecalibratedasdescribedinthetext.
Thecis/trans-isom-erizationofPro2andPro4residuesinsubPhasasignificantinfluenceon[subP+3H]3+conformationsandplaysakeyroleinmanyofthetransitionsobserved.
Comparisonsofstructuralchangesanddissociationpatternsinthegasphasetothosefromsolutionshowthatsolventplaysakeyroleinregulatingcon-formationsinsolution.
AcknowledgementsThisworkissupportedinpartbyfundsfromtheNationalInstituteofHealth,R01GM117207-04,andtheIndianaUni-versityRobertandMarjorieMannfellowships(CRC,DRF).
TheworkatTAMU(DHR)wasfundedbyNSF(CHE-1707675)andNIH(P41GM121751-01A1).
C.
R.
Conantetal.
:SubstancePintheGasPhase:ConformationalChangesandDissociations943944C.
R.
Conantetal.
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