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volume19number12013ISSN1469-0667INTHISISSUEhumanplacentatissuebymatrix-assistedlaserdesorptionionisationmatricesfornegativemodemetabolomicselectricalconductivityofmatrixcrystals EUROPEANJOURNALOFMASSSPECTROMETRYEditor-in-ChiefPeterJ.DerrickInstituteofFundamentalSciencesMasseyUniversityPalmerstonNorthCampusPrivateBag11222PalmerstonNorth5301NewZealand.E-mailp.j.derrickmassey.ac.nz.EditorsEditorialAdvisoryBoardProfessorJrgenGrotemeyerInstittfrPhysikalischeChemieChristian-Albrechts-UniversittzuKielLudewig-Meyn-Strae824098KielGermanyFax494318802828E-mailgrotephc.uni-kiel.deProfessorMichisatoToyodaProjectResearchCenterforFundamentalSciencesGraduateSchoolofScienceOsakaUniversity1-1MachikaneyamaToyonakaOsaka560-0043Japan.Fax81-6-6850-5762E-mailtoyodamphys.sci.osaka-u.ac.jpProfessorChrysWesdemiotisDepartmentofChemistryTheUniversityofAkronAkronOH44325-3601USA.Fax1-330-972-7370E-mailwesdemiotisuakron.eduProfessorVladimirZaikinTopchievInstituteofPetrochemicalSynthesisRussianAcademyofSciencesLeninskyProspect29117912-GSPMoscowRussia.Fax7-095-2302224E-mailzaikinips.ac.ruProtocolsEditorProfessorDrJonasBergquistDepartmentofChemistryBMCAnalyticalChemistryUniversityofUppsalaUppsala745124Sweden.E-mailjonas.bergquistkemi.uu.seBookReviewEditorProfessorO.DavidSparkman5354ThunderbirdCt.AntiochCA94531USA.E-mailodscompuserve.comJ.AmsterAthensGAA.AshcroftLeedsU.BoeslMunichR.D.BowenBradfordK.BreukerInnsbruckT.-W.D.ChanCUHKHKY.-J.ChenTaipeiJ.J.CoonMadisonWIC.E.CostelloBostonMAC.S.CreaserLoughboroughK.DownardSydneyT.DrewelloErlangenJ.H.FutrellRichlandWAA.GiannakopulosBremenM.GlockerRostockF.GrandinettiViterboS.HammerumCopenhagenV.HavlicekPragueA.J.R.HeckUtrechtO.LaprvoteGif-sur-YvetteA.LebedevMoscowC.B.LebrillaDavisCaliforniaA.MalletGreenwichA.MikaiaGaithersburgMAE.NikolaevMoscowR.A.J.OHairMelbourneL.RadomCanberraG.E.ReidEastLansingMIB.SpenglerGiessenJ.K.TerlouwHamiltonONJ.F.J.ToddKentP.TraldiPaduaY.O.TsybinLausanneM.VairamaniChennaiP.WilliamsTempeAZR.ZenobiETHZrich EUROPEANJOURNALOFMASSSPECTROMETRYAnasteriskagainstanauthorsnameonthefrstpageoanarticleindicatestheauthortowhomcorrespondenceshouldbeaddressed.ContentsQualitativecharacterizationoDesmodiumadscendensconstituentsbyhigh-perormanceliquidchromatography-diodearrayultraviolet-electrosprayionizationmultistagemassspectrometry1ClaudioBaiocchiClaudioMedanaValeriaGiancottiRiccardoAigottiFredericaDalBelloCristinaMassolinoDanielaGastaldiandMaurizioGrandiDetectionotheepidermalgrowthactorreceptoramphiregulinandepiregulininormalin-xedparan-embeddedhumanplacentatissuebymatrix-assistedlaserdesorptionionizationmassspectrometryimaging17KhaledMahmoudLauraM.ColeJillianNewtonSabahMohamedMaherAl-EnaziPhilQuirkeandMalcolmRClenchAthiophene-containingcompoundasamatrixormatrix-assistedlaserdesorptionionizationmassspectrometryandtheelectricalconductivityomatrixcrystals29AkikazuYasudaTakayukiIshimaruShogoNishiharaMasamichiSakaiHideyaKawasakiRyuichiArakawaandYasushiShigeriMatrix-assistedlaserdesorptionionizationmatricesornegativemodemetabolomics39StephanR.FagererSimoneNielsenAlredoIbezandRenatoZenobiFragmentationcharacterizationanddierentiationoisomericdiglycosylfavonoidsusingultra-high-perormanceliquidchromatographyelectrosprayionizationquadrupoletime-o-fighttandemmassspectrometryinnegativeionmode49XueengGuoYongdeYueFengTangJiaSunJinWangXiYaoandHangXunAtemplateapproachorthecharacterizationolinearpolyaminesandderivativesinspidervenom57ManuelTzourosSergeChesnovLaurentBiglerandSteanBienzLetterMassspectrometricapproachohighpH-andcopper-inducedglutathioneoxidation71GabiDrochioiuLauraIonCatalinaCiobanuLauraHabasescuandIonelMangalagiu IMPublicationsLLP6CharltonMillCharltonChichesterWestSussexPO180HYUK.Tel44-01243-811334Fax44-01243-811711WebSitewww.impublications.comHowtosubmityourpaperYoucansubmityourpapereithertoouronlinemanuscriptsubmissionandtrackingsystematwww.impublications.comeauthorsordirecttoIMPublications.PleasesendmanuscriptstoGillStockordIMPublicationsLLP6CharltonMillCharltonChichesterWestSussexPO180HYUKE-mailgillimpublications.co.uk.PleasedoNOTsendyourmanuscripttooneotheEditors.InstructionsorAuthorsappearatthebackomostissuesothejournalandmaybeobtainedromthePublishersorourwebsitewww.impublications.comejms.OnlinemanuscriptsubmissionTheonlinemanuscriptsubmissiontrackingandreviewsystemislocatedatwww.impublications.comeauthors.Youwillneedtoregisterbeoreyoucanusethesystem.OnceregisteredloginandclickontheAuthorlinkunderthejournaltitle.FromhereclickonthelinktoStarttheSubmissionProcessandollowtheinstructions.Thesystemsavesyoursubmissionasgoyougothroughthefve-stepprocesssoyoucanreturntoitiyouareinterrupted.Onceyouhavecompletedasubmissionyouwillbeabletoviewinormationontheprogressoyourpapersthroughthepublicationprocess.Iyouhaveanyproblemsorqueriespleasecontactusorhelp.HelpustopublishyourpaperquicklyOurproductionprocessisentirelydigitalandwewouldliketoreceiveyourmanuscriptinelectronicormweusethelatestversionoWordorWindowswhichcanreadmostotherormatsiindoubtuseRTF.Wewillnormallye-mailyourproostoyouasanAdobeAcrobatPDFflesopleasemakesureyouprovideyoure-mailaddress.EUROPEANJOURNALOFMASSSPECTROMETRYManagingBoardChairmanProfessorHans-FriedrichGrtzmacherFakulttrChemieUniversittBieleeldPostach100131D-33501BieleeldGermany.Fax49-521-106-6417e-mailgruetzmacherchema.uni-bieleeld.deProfessorJ.H.BowieOrganicChemistryDepartmentG.M.BadgerLaboratoriesTheUniversityoAdelaideAdelaideSouthAustralia5001Australia.Fax61-8-224-0464e-mailjbowiechemistry.adelaide.edu.auProfessorLeopoldoCerauloUniversitadegliStudidiPalermoDipartimentodiChimicaeTecnologieFarmaceuticheLaboratoriodiSpettrometriadiMassaViaArchiraf32I-90123PalermoItaly.Fax39-091-6236110e-maillceraulounipa.itProfessorJohnL.HolmesDepartmentoChemistryUniversityoOttawa10MarieCuriePOBox450StnAOttawaONK1N6N5Canada.Fax1-613-562-5170e-mailjohnleonard.holmesuottawa.caProfessorMamoruhashiDepartmentoChemistryKanagawaUniversityTsuchiyaHiratsukaKanagawa259-1293Japan.Fax81-463-58-9684e-mailohasimchem.kanagawa-u.ac.jpProfessorJean-ClaudePromLaboratoiredesIMRCPUMR5623UniversitPaulSabatier118RoutedeNarbonne31062ToulouseCedexFrance.E-mailpromeramses.univ-tlse.rProfessorDouglasP.RidgeDepartmentoChemistryandBiochemistryUniversityoDelawareNewarkDelaware19716USA.Fax1302831-6335e-maildougrudel.eduProfessorEinarUggerudKjemiskInstituttUniversitetetiOsloPostboks1033BlindernN-0315OsloNorway.Fax47-22855441e-maileinar.uggerudkjemi.uio.no ISSN1469-0667IMPublicationsLLP6CharltonMillCharltonChichesterWestSussexPO180HYUK.Tel44-01243-811334Fax44-01243-811711WebSitewww.impublications.comAimsandscopeEuropeanJournalofMassSpectrometryEJMSisdevotedtotherapidpublicationooriginalresearchpapersconcernedwiththemassspectrometryobiologicalinorganicandorganicsamples.Thescopeothejournalencompassesmolecularionisationandragmentationgas-phaseionchemistryionmoleculereactionsandcollisionsspectroscopyogaseousionsinstrumentationcomputersinmassspectrometryandoriginalapplicationsintheliesciencesenvironmentalscienceandindustry.Topicsointerestincludeexperimentalandtheoreticalstudiesostructuresenergeticsandreac-tionsogaseousionsionormationrombiologicalsolidsdeterminationostructureobiomoleculesmechanismsoionicreactionsionisationphenomenaelectrosprayionisationESImatrix-assistedlaserdesorptionionisationMALDImassspectrometryopolymersreactionsometalionsreac-tionsoionsatsuracesmulti-photonionisationanddissociationtime-o-fightTOFtandemmassspectrometryFouriertransormioncyclotronresonanceFT-ICRandtheoriesoionormationstruc-turesandreactions.IMPublicationsLLP2013Allrightsreserved.Apartromanyairdealingorthepurposesoresearchorprivatestudyorcriti-cismorreviewaspermittedundertheUKsCopyrightDesignsandPatentsAct1988thispublicationmaybereproducedstoredortransmittedinanyormorbyanymeansonlywiththepriorpermissioninwritingothepublishersorinthecaseoreprographicreproductioninaccordancewiththetermsolicencesissuedbytheCopyrightLicensingAgency.Enquiriesconcerningreproductionoutsidethosetermsshouldbesenttothepublishersattheaddressabove.SubscriptiondetailsEuropeanJournalofMassSpectrometryispublishedassixissuesperVolume.Thesubscriptionratesare587.00deliveryaddressesinEurope741.00deliveryaddressesinEuropeorUS966.00deliveryaddressesoutsideEuropeorVolume192013andthisincludesaccesstothewebeditionorallonasubscribingsite.Web-onlysubscriptionsareavailableatreducedratescontactthepublishersormoreinormation.SubscriptionscanonlybeacceptedoraullcalendaryearandshouldbeorderedromthepublishersIMPublicationsLLP6CharltonMillCharltonChichesterWestSussexPO180HYUKTel4401243811334Fax4401243811711orromyoursubscriptionagent.ThelatestsubscriptioninormationisalwaysavailableontheEJMSWebSiteURLhttpwww.impublica-tions.comejms.MembersotheBritishMassSpectrometrySocietytheDeutscheGesellschatrMassenspektrometrietheSocitFranaisedeSpectromtriedeMasseandtheSocietChimicaItalianacanbenetromreducedratesubscriptionscontactyoursocietysadministrationordetails.AbstractingEuropeanJournalofMassSpectrometryisabstractedinAnalyticalAbstractsBIOSISChemicalAbstractsChemistryCitationIndexCurrentContentsPhysicalChemicalEarthSciencesMassSpectrometryBulletinResearchAlertSciSearch.InadditionabstractsareavailableontheEJMSWebSiteURLhttpwww.impublications.comejmsinadvanceopublication.Otherabstractingpublicationsinter-estedinincludingEJMSareinvitedtoapplytothePublisher.AdvertisingAdvertisingisacceptedinEuropeanJournalofMassSpectrometryurtherdetailscanbeobtainedromtheAdvertisementManagerIMPublicationsLLP6CharltonMillCharltonChichesterWestSussexPO180HYUKTel4401243-811334Fax4401243-811711E-mailadsimpublications.co.uk.PrintedintheUKbyLatimerTrendCompanyLtdPlymouth. 1ISSN1469-0667IMPublicationsLLP2013doi10.1255ejms.1214AllrightsreservedEUROPEANJOURNALOFMASSSPECTROMETRYDesmodiumadscendensDASwD.C.var.AdscendensPapilionaceaeisamedicinalherbcommonlyusedinAfricantraditionalmedicine.Itisreportedtobeusedforasthmatoaidparturitiontotreatdysmenorrhoeaandtoimprovelactation.Itisknowntohavehepato-protectivepropertiesandisemployedtomanageseveralotherconditionsincludingfeverpainandepilepsy.Itisatypicalexampleofasingleplantbeingusedformanyapparentlyunrelateddiseases.1ManystudieshavereportedthatDesmodiumadscendensactivatescalcium-dependentpotassiumchannels2inhibitsNADPH-dependentQualitativecharacterizationoDesmodiumadscendensconstituentsbyhigh-perormanceliquidchromatography-diodearrayultraviolet-electrosprayionizationmultistagemassspectrometryClaudioBaiocchiaClaudioMedanaaValeriaGiancottiaRiccardoAigottiaFredericaDalBelloaCristinaMassolinoaDanielaGastaldiaandMaurizioGrandibaUniversityofTorinoDepartmentofMolecularBiotechnologyandHealthSciencesviaP.Giuria510125TorinoItaly.E-mailclaudio.medanaunito.itbPolyclinicLaTorreviaM.Ponzio1010141TorinoItalyThemanyeectsotheAricanmedicinalherbDesmodiumadscendenswerestudiedinthe1980sand1990s.Inspiteothisacomprehensiveanalyticalprotocolorthequalitycontroloitsconstituentssoyasaponinsalkaloidsandfavonoidshasnotyetbeenormulatedandreported.ThisstudydealswiththeoptimizationoextractionconditionsromtheplantandqualitativeidenticationotheconstituentsbyHPLCdiodearrayUVandmultistagemassspectrometry.Plantconstituentswereextractedromleavesbyliquidliquidandsolidmatrixdispersionextraction.SeparationwasachievedviaRP-C18liquidchromatographywithUVandMSndetectionandmassspectrometryanalysiswasconductedbyelectrosprayionizationiontrapororbitrapmassspectrometry.HighresolutionmassspectrometryHRMSwasusedorstructuralidenticationoactivemoleculesrelatingtosoyasaponinsandalkaloids.ThefavonoidragmentationswerepreliminarilystudiedbyHRMSinordertoaccuratelycharacterizethemorecommonneutrallosses.Howeverthehighnumberoisomericspeciesinducedustomakerecoursetoamoreextendedchromatographicseparationinordertoenableuseultandemmassspectrometryandultravioletspectralinterpretationtoproposeareasonablechemicalclassicationothesepolyphenols.35compoundsothisclasswereidentiedhereinwithrespecttothevereportedinliterature.Inthiswaywemadeupacomprehensiveprotocolorthequalitativeanalysisothehighcomplexitycontentothisplant.Thisresultpavesthewayorbothreliablequalitycontrolopotentialphytochemicalmedicamentsandpossibleuturesystematicclinicalstudies.KeywordsDesmodiumadscendenssoyasaponinsfavonoidsDADUVHRMSIntroductionC.Baiocchietal.Eur.J.MassSpectrom.191152013Received31July2012nRevised27February2013nAccepted13March2013nPublication17April2013ejmsprotocols 2QualitativeCharacterizationofDesmodiumadscendensConstituentsoxygenationoarachidonicacid3actsasananti-anaphylactic3andisaninhibitorohistaminerelease.4AcompletereviewothepharmacologicalactionsoDesmodiumadscendenshasbeenreported.5Theactiveingredientsotheplantaresoyasaponinsfavonoidsphenolicandsimpleheterocyclicalkaloids.Theywereisolatedandidentiedduringthe1990sinparticularsoyasapogenolBandE6soyasaponinIdehydrosoyasaponinIandsoyasaponinIII.3Thefavonoidsidentiedweretecto-rigenineapigenine-7-O-glucosideandkaemperol-3-O-glu-coside.7Alkaloidswhichareminorconstituentsotheplantwerealreadyknowninthe1980sandthelisthassincebeenupdated.89SoyasaponinsandalkaloidsstructuresarereportedinFigure1.Atpresentthereisalackoanalyticalmethodsabletocharacterizeallthedierentclassesoplantcomponentseitherqualitativelyorquantitatively.Specicextractionandpuricationmethodsorobtainingpurecompoundstostudypharmacologicalpropertieshavebeenreported911howeverthesearelimitedtosomeothesoyasaponins.MorerecentlyaplanarchromatographicstudytovalidateingerprintsoDesmodiumadscendenswaspublished12butitdoesnotsupplyinormationaboutthepresenceoalkaloids.Therelativelylowsensitivityothetechniqueleadstothecategorizationosoyasaponinsasthemainuseulmarkersotheplantandundervaluatingtheroleofavonoidsonlyvitexineisovitexineandquercetinerutinosidewereidentied.Inthepresentstudyhigh-perormanceliquidchromato-graphyHPLCseparationoDesmodiumadscendensextractsromsolid-phasematrixdispersionextractionSPMDEtreat-mentotheleaveswasperormedwithveryreliabledetectionusingUV-Diodearrayandiontrapmassanalyzerdetection.Structuraldetailswereobtainedbytandemmassspectrom-etryandidenticationwasurthersupportedbyhighreso-lutionmassspectrometryHRMSanalysis.HighresolutionmassspectrometerseatureswereextensivelyreviewedbyMarshallandHendrickson13aewyearsago.Theperor-mancesandcapabilitiesoorbitrapmassanalyzerweredescribedinanumberoreviewarticlesespeciallyintheeldobioanalysis.1415TheseauthorsemphasizethatLC-MSmethodsbasedonorbitrapaccuratemassmeasurementsrepresenttodayanindispensableanalyticaltoolorthenon-targetscreeningandstructuralelucidationometabolitesduetoull-scansensitivityhighselectivityandspecicityincontrasttominordrawbackslikepoorMS2sensitivityinthecaseotime-o-fightToFandhighcostinthecaseoioncyclotronresonanceICR.Inparticularweidentiedinthefavonoidclassmanymoreconstituentsthanhadpreviouslybeenreported.ThestudyothisclassocompoundscouldnotrelyonHRMSinormationduetothelargenumberoisobaricsubstancesundistinguishablebytheirmzvalues.Themanycomponentsarecomprisedovariously-glycosylatedormsessentiallyotwoaglyconesapigenineandkaemperolalthoughdiosmetin4-methoxyapigenineisalsopresent.ThisindicatesthattheremustbeO-glycosylationandorC-glycosylationatdierentsubstitutionpositionsandindierentcombinations.OnthecontraryspectrophotometricinormationromdiodearraydetectorDADwasoparticularimportanceinsupportingthedeterminationocompoundsbelongingtothechemicalclassofavonoids.Thein-depthinterpretationothelow-resolutionMSMSspectraenabledustocharacterizenumerouscompounds.Inordertoutilizethesetwodetec-tiontechniquestomakereliableidentiicationsthechro-matographicseparationolavonoidshadtoprovidegoodresolution.Theoptimizationotheanalyticalcharacteriza-tionperormedtoalloworuturestudiesofavonoidsasmoleculeswithinterestingpharmacologicalpropertieseitherdirectlyorsynergistically.SOYASAPONINIIIC42H68O14796.461RRha-GlcUAOHROOHSOYASAPOGENOLBC30H50O3458.375OHHOOHRGlc-Rha-GlcUARGlc-Rha-GlcUASOYASAPONINIDEHYDROSOYASAPONINIC48H76O18940.503OHROOC48H79O18943.526OHROOHNHOC10H16NO165.123HORDENINNHHOCH3OC11H16NO2193.118SALSOLINENHNC12H17N2188.139DIMETHYLTRIPTAMINECH3OOCH3NH2C10H16NO2181.118DIMETHOXYPHENYLETHYLAMINEFigure1.StructuresofDesmodiumadscendenssoyasaponinstopandalkaloidsbottom. C.Baiocchietal.Eur.J.MassSpectrom.1911520133InparticularC-glycosidesmayplayanimportantroleinprovidingDesmodiumadscendenspropertiesasanutraceu-ticalproduct.IngeneralvariousbiologicalactivitieshavebeenreportedorfavoneC-glycosidesincludingtheantimicrobialactivityovitexineandthehepatoprotectiveeectoluteoline-7-glucosidetogetherwiththeantioxidantactivitythatischar-acteristicotheentireclassofavonoids.Quantitativeevaluationothevariousconstituentswasimpos-sibleduetothelackocommercially-availablepurestandardsandthereoreourresultsareonlyqualitative.Howevertheinstru-mentalcongurationadoptedopensthedoortouturestudiesocomparativequalitycontrolandtheconstituentsdistributionbasedongeographicorigin.Furthermoretheresultingknowl-edgeothechromatographicbehaviorothecomponentsidenti-edallowsHPLCpuricationoeitherasinglesubstanceoranentireclassosubstancesthuspavingthewaytomoreaccuratestudiesothepharmacologicaleectsothisplant.Theexpensiveinstrumentalcongurationusedinthisstudywasuseulinunderstandingthetypicaldistributionofavo-noidsinDesmodiumadscendensthatmakesthemreliablemarkersotheplantasimplerdetectorcouldbeusedtodevelopqualitycontrolanalyticalmethods.ExperimentalMaterialsandreagentsPlantmaterialleaveswasprovidedbyAVDReormParmaItaly.AllextractionandchromatographicsolventswereHPLCgradeVWRMerckDarmstadtGermany.HPLCgradewaterwasromMilliQSystemAcademicMilliporeMilanItaly.Octadecyl-unctionalizedsilicagelandtyraminewereromSigma-AldrichMilanItaly.Vitexinsaponarineapigenin-8-C-7-O-diglucosidevitexin-2-O-rhamnosideandapigenine-7-O-glucosidewereromExtrasynthseGenayFrance.SamplepreparationTwodierentextractionprocedureswereusedorsoyasaponinsandlavonoids.TheSPMDEtechniquewastestedtoextractractionsodierentpolarityromDesmodiumadscendensleaves.Theplantleaveswerecrushedtoanepowderusingamilland2.5gothepowderwerecareullymixedwith2.5gooctadecyl-unctionalizedsilicagel200400mesh.Solventsodecreasingpolarityweresuccessivelyelutedinorderwatermethanolmethylenechlorideandn-hexaneorextractionosoyasaponinsandfavonoids.Themoreclassicsoxhletextractionwithethanolwater7030ortwodayswastestedasanalternativewiththeaimoobtainingalessspecicbutmorecompleterecoveryoallconstituents.Toevaluateextractionmethodswecomparedpeakareavaluesothevariousconstituents.Astandardmixtureotyraminevitexinandapigenine-7-O-glucosidewasusedasasemi-quantitativereerence.Suchvalueswerenotshowingsignicantdierences.HoweverinbothextractionmethodsalkaloidswereonlyrecoveredinsmallamountssoaspecicliquidliquidprocedureollowedbyanSPEpuricationstepwasusedorthisamilydrypowder0.2gwassetina15.0mLtubecontaining10.0mLo1HClandkeptatroomtemperaturewithoccasionalshaking.Aterthreetoourdaysextractionthesamplewascentriugedat3000rpmor15minat4C.A3.0mLsolid-phaseextractionaromaticsulonicacidcolumnVarianBondElutCertiyIIVarianLeinItalycontaining500.0mgopackingmate-rialwaspreparedbywashingwith3.0mL80methanoland3.0mLdistilledwaterunderamoderatevacuumaterwhichthesamplesupernatantwasloadedontothecolumntoadsorbalkaloids.Thecolumnwasrinsedwith23mLeacho1HCldistilledwaterandmethanol.Alkaloidswereelutedusing2.0mLethanol10NH4OHinmethanol11vvsolution.ThesolventwasevaporatedimmediatelyunderN2gasfowandtheresiduedissolvedin50Lomethanol.High-performanceliquidchromatographyultraviolet-electrosprayionizationtandemmassspectrometryconditionsThedierentsetupsusedconsistedoa1100AgilentHPLCsystemwithadoubledinseriesdetectionsystemsaphotodiode-arraydetectorandaXCTPlusiontrapmassspectrometerAgilentMilanItalyandanUltimate3000HPLCsystemDionexMilanItalyequippedwithanon-lineDADUVdetectorandaLTQ-Orbitraphighresolutionmassspectrom-eterThermoScienticBremenGermanyrespectively.BothsystemswereequippedwithanelectrosprayESIinterace.DesmodiumadscendensconstituentswereseparatedonaPhenomenexCastelMaggioreBOItalyLunaC18column1502.0mm3mparticlesizeelutedindierentgradientelutionconditionsdependingontheclassocompoundstobeseparated.Thefowratewasinallcases0.25mLmin1andthesampleinjectionvolumewas20L.Foralkaloidsthemobilephasewascomprisedotwosolventsheptafuorobutanoicacid5.0mMinwatersolventAandacetonitrilesolventBinalineargradientprogramrom937ABto2971in21min.Forsoyasaponinsthemobilephaseconsistedotwosolventsaceticacidinwater0.599.5vvsolventAandaceticacidinacetonitrile0.599.5vvsolventBinagradientprogramwhichwasinitially8812ABandgradedrom0to100in38min.Inthecaseofavonoidsthemobilephasecomprisedthesamesolventsasorsoyasaponinsseparation.Initialelutionconditionswere9010ABat36minthesewerechangedto8812at61minto8515at71minto8218andat85mintheywerechangedto7030.Thesourcevoltagewassetto4.2kV.Theheatedcapillarytemperaturewasmaintainedat275C.Theacquisitionmethodusedhadpreviouslybeenoptimizedortheparentcompoundcapillarymagneticlensesandcollimatingoctapolevoltagesinordertoachievemaximumsensitivity.Themaintuningparam-etersadoptedortheESIsourcewere7.00Vorthecapillaryvoltageand80Vorthetubelens.Fullscanspectrawereacquiredintherange501000mz.MSnspectrawereacquiredintherangebetweeniontrapcut-oandprecursorionmzvalues.High-resolutionmassaccuracyorecordedionsvscalculatedwas 4QualitativeCharacterizationofDesmodiumadscendensConstituents5millimassunitswithoutinternalcalibration.High-resolutionspectrawereacquiredwiththeresolutionR30000FWHM.ResultsanddiscussionOnlythemethanolicractionromSPMDEexhibitedasignicantpresenceosoyasaponinsandfavonoids.HPLC-photodiodearray-MSanalysisothen-hexanemethylenechlorideandwaterelutionractionsshowedonlytracessignals.Alkaloidswererecoveredonlybyalkalineextractionconditions.SoyasaponinsandalkaloidscharacterizationSoyasaponinswereidentiiedandcharacterizedbyhighresolutionmassspectrometryinullmasspositiveionmodeandinMSnmode.Figure2showsthechromatographicsepa-rationosoyasaponinsrecognizedbytheirMHmzvalues.IdentiicationwasconirmedbyMSMSexperimentsandbyaccuratemassdeterminationoprecursorandproductions.SoyasaponinIdehydrosoyasaponinIsoyasapogenolBandsoyasaponinIIIwereidentied.Table1summarizesESI-orbitrapMS2accuratemassdata.Theragmentationpathwaysothesoyasaponinsaredominatedbythestep-by-stepeliminationosugarmoietiesandlossesowatermolecules.AsanexampletheMS2spectrumosoyasaponinIisreportedinFigure3togetherwiththerelativeragmentationpathways.AlkaloidsareminorconstituentsoDesmodiumadscendensandconditionstoachievetheirextractionareverydemandingseeExperimentalsection.Figure1showsthestructuresotheouralkaloidstypicalotheplantidentiedinthealkalineextract.ThemzvaluesoprecursorandmoreintenseproductionsarereportedinTable1.Theirragmentationpathwaysproceedbythelossoammoniaoraliphaticamineswhenaethylaminomoietyispresentthemethyl-tetrahydro-isoquinolinicderivativesalsolinelosesamethylradicalwithormationotheM15oddelectronproduction.Their02468101214161820222426283032343638404244464850Timemin020406080100RelativeAbundance020406080100RelativeAbundance020406080100RelativeAbundance020406080100RelativeAbundance41.827.628.926.6Figure2.Soyasaponinschromatographicseparation.FromtoptobottomsoyasapogenolBsoyasaponinsIIIdehydrosoyasaponinIandsoyasaponinI.Experimentaldetailsaredescribedinmaterialandmethodssection. C.Baiocchietal.Eur.J.MassSpectrom.1911520135CompoundMHMS2SoyasaponinI944.534797.456617.395441.366599.385SoyasaponinIII797.468599.397411.373599.195SoyasapogenolB459.383441.373423.363Dehydro-soyasaponinI941.510795.453439.358615.390Hordenine166.130121.065C8H9O107.049C7H7ODimethyltriptamine189.139130.065C9H8N84.081C5H10NDimethyoxyphenylethylamine182.118165.091C10H13O2137.060C8H9O2Salsoline194.118179.094C10H13NO2Table1.ESIHRMS2massdataforsoyasaponinsandalkaloids.OHOOHOCOOHOHHOOOOHHOHOOOHOOHOHC48H80O18944.534OHOOHOCOOHOHHOOOOHHOHOOHOHOOHOHHC42H69O14797.456OHOOHOCOOHOHHOOHOOHHOHOOHHC36H59O9635.406OHHOOHOCOOHOHHOOHHHC30H51O3459.384-H2OHOHOHOHHH-H2O-H2OC36H57O8617.395-H2O-H2O-H2OC36H55O7599.395C36H53O6581.384C30H49O2441.397C30H47O423.356C30H45405.345Figure3.MS2spectrumofsoyasaponinIprotonatedprecursorionatmz943.526andrelativeproposedfragmentationpathways.Precursorandproductionsweremonitoredinhighresolutionmode. 6QualitativeCharacterizationoDesmodiumadscendensConstituentsconcentrationsevaluatedusingtyramineassemi-quantitationstandardcompoundwereunder0.1otheplantmaterial.FlavonoidscharacterizationThedistributionolavonoidsinDesmodiumadscendensisparticularanduseultocharacterizetheplantmaterial.WithregardtoglycosydiccompoundsromMSnanalysisitwaspossibletorecognizetheelementalcompositionotheaglyconewhichwasidentiableromtheUVspectrum.MassspectraandUVabsorptiondatashowthatmainlyderivativesofavoneapigeninandfavonolkaemperolarepresentinsigni-icantamountsalongwithsmallquantitiesodiosmetinwhichishoweveramethoxyderivativeoapigenin.Aquercetinederivativehasalsobeenidentied.Themainfavonoidconstit-uentsidentiedrangingrom1to20inrespecttototalarelistedinTable2.WereportedretentiontimesmzvalueoMHcorrespondingionsaglyconesglycosylationpositionsandasemiquantitativeestimationdoneonthebasisopeakareasrelativetothatotheglycosylatedfavonesvitexineasastandardcompound.TheuseoamassspectrometerdetectoringeneralmeansthatgoodchromatographicseparationconditionscanbeRetentiontimeMHCompositionRelativeamount10.76116C8C-dihexosyl-kaemperol12.34335-O-hexosyl-apigenin18.15956-C8-C-dihexosyl-apigenin20.55816-C-pentosyl-8-C-hexosyl-kaemperol22.55816-C-hexosyl-8-C-pentosyl-kaemperol24.54495-O-hexosyl-kaemperol25.25956-C-hexosyl-8-C-pentosyl-diosmetin25.65816-C-pentosyl-8-C-hexosyl-kaemperol27.75816-C-hexosyl-8-C-pentosyl-kaemperol27.95656-C-pentosyl-8-C-hexosyl-apigenin31.94498-C-hexosyl-kaemperol32.15816-C-pentosyl-8-C-hexosyl-kaemperol35.55656-C-pentosyl-8-C-hexosyl-apigenin38.65816-C-hexosyl-8-C-rhamnosyl-kaemperol44.35656-C-hexosyl-8-C-pentosyl-apigenin44.85815-O-pentosyl-16-rhamnosyl-kaemperol46.4595Saponarin6-C-hexosyl-7-O-hexosyl-apigenine47.15817-O-pentosyl-16-rhamnosyl-kaemperol48.65656-C-hexosyl-8-C-pentosyl-apigenin52.5433Vitexin8-C-hexosyl-apigenin53.25795-O-rhamnosyl-1-6-hexosyl-apigenin54.55655-O-pentosyl-1-6-hexosyl-apigenin54.65816-C-hexosyl-8-C-pentosyl-kaemperol57.9449Astragalin3-O-hexosyl-kaemperol61.75796-C-hexosyl-8-C-rhamnosyl-apigenin62.35955-O-pentosyl-16-hexosyl-diosmetin64.95656-C-hexosyl-8-C-pentosyl-apigenin65.25796-C-rhamnosyl-8-C-hexosyl-apigenin67.85796-C-hexosyl-7-O-rhamnosyl-apigenin70.24477-O-rhamnosil-quercetin71.25796-C-rhamnosyl-8-C-hexosyl-apigenin72.54497-O-hexosyl-kaemperol74.757916-rhamnosyl-7-O-hexosyl-7-apigenin76.14337-O-hexosyl-apigenin78.15957-O-pentosyl-16-hexosyl-diosmetinTable2.ListomainconstituentsoDesmodiumadscendensinthefavonoidclass. C.Baiocchietal.Eur.J.MassSpectrom.1911520137letoutoconsideration.HoweverinthepresentcasethelargenumberoisomericormsandtheneedorsupportinginormationromUVabsorptionspectraorcedustoexploredierentchromatographicconditionstheaimwastooptimizetheseparationoalllavonoidconstituentsoDesmodiumadscendens.Aterseveralattemptsweobtainedasatisactoryseparationothemajorityoconstituentspresentinsignicantamountsalongwiththeirclearly-denedUVspectra.Figure4showsthechromatogramoamethanolicextractoDesmodiumadscendensmonitoredbyUVdetectionatlo330nm.Ascanbeseentheoverallanalysistimewasabout90minutesandthenumberoconstituentswashuge.ThekaemperolorapigeninederivativesdistinguishedromtheirUVspectraandaglyconemoietyidenticationbyMSnareindicatedbysubscriptsKorA.TheMSMSdataallowedspeculationabouttheglycosylationothetwofavonoids.WecarriedouttheragmentationstudybothbyiontrapMSnandbyHRMSn.Thisissuelooksmorecomplexbecauseothepresenceoseveralisomericormsduersttocombinationsodistinctaglyconesdivergentby16Dawithdierentsugarsdieringby16Daasglucoseandrhamnosebutalsotodierentglycosylationpositionsothesamesugars.Consequentlytherelativeretentiontimesareaectedbythetypeandnumberolinkedsugarsurthermoreaglyconeandsugarbeingequaltheyarealsoaectedbythedierentglycosylationpositions.ExaminingthisquestioningreaterdepthmorethanonespeciescorrespondstoeachtypeomonoglycosidedependingonwhetheritisanO-glycosideoraC-glycosidederivative.ThereisnodierenceinUVspectranorinthemzvalueotheprecursorionbutthereisadierenceinMSMSspectra.MoreoverinbothpositiveandnegativeionmodesO-glycosidederivativesgiveverydierentMSMSspectraromC-glycosidederivatives.InthepositiveionmodeO-glycosidederivativesshowthelossothesugarmoietyasthemainragmentationpathway.ConverselyC-glycosylderivativesshowcross-ringcleavagesothesaccharideresidueandsuccessivelossesomoleculesowaterandormaldehyde.AsexpectedthemuchgreaterstrengthotheC-sugarbondversustheO-sugarbondallowsotherragmentationpathwaystooccurinvolvingthehydroxylgroupsothesugarcyclizationprocessesthusbecomeenergeticallycompetitive.Severalsimilarsituationshavebeenreported.16Figures5aandbshowthetypicalMSMSspectraotheO-glycosylatedapigenineandthecorre-spondingC-glycosylatedorm.AllthefavonoidspectrawerereplicatedbyHRMSnanalysistoconrmunambiguouslytheneutrallosseshypothesized.Thequestionoinwhichpositiontheglycosylationoccursisdiiculttodetermineandmassspectraldataareolimiteduseulness.InthecaseoO-glycosylationthemassspectraldatadonotprovidethepositionalinormation.InthecaseoC-glycosylationandoCC-diglycosylationoverallitmayonlybededucedromthedierenceininten-sityocertainmzvaluespresentinthemassspectraobtainedineitherthenegativeorthepositiveionmode.AsanexampletwopositiveionMSMSspectraareshowninFigures5canddwithregardtotwodiglycosidederiva-tivesoapigeninewitharabinoseandglucosealternatelyintheC-6andC-8positions.Severalquantitativedier-encesinmzvaluedistributionmaybeseen.InthecaseoglucoseatC-8theionsormedbywaterlossaremoreKkaempferolAapigenin0100200300400500mAU1020304050607080TimeminKAAAAAAAAAKKKKKKKKAKAKAAwavelengthnmRelativeAbsorbance336270230wavelengthnmRelativeAbsorbance22634827076583OOHOOHHOOHACB76583OOHOOHHOACBKAFigure4.UVchromatogramofavonoidractionmonitoredatlo330nm.UVspectrumokaemperoltopboxandapigeninbottombox.Kaemperol-relatedspectraaremarkedwithKandapigenin-relatedwithA. 8QualitativeCharacterizationoDesmodiumadscendensConstituentsFigure5.MS2spectratogetherwithproposedhypotheticalragmentationpathwaysotheO-glycosilatedapigeninaandthecorrespondingC-glycosylatedormb.MS2spectraotwodierentprecursorionsat565mzwithaglyconeat271mzcandd.NeutrallossidentitywasconfrmedbyHRMSn.140180220260300340380420460mz0102030405060708090100RelativeAbundance271140180220260300340380420460mz0102030405060708090100RelativeAbundance415397364313379337ABC19H13O6337mzC21H15O7379mzC20H15O7367mzCH2OCH2OH2OC21H19O9415mzH2O8OOHHOOHOHHO8OOHHOOHOHOOHOHHOHOPIGENIN-8-C-GLUCOSIDEC21H21O10433mzC17H13O6313mzC4H8O4120.042OOHOHHOHO7OOHOOHOHAPIGENIN-7-O-GLUCOSIDEC21H21O10433mzC15H11O5271mzOOHOHOHOHC6H10O5162.053160200240280320360400440480520560600mz5475294275110102030405060708090100RelativeAbundanceC160200240280320360400440480520560600mz5474995294574814694455110102030405060708090100RelativeAbundanceDOHOHOOH68OOHOHHOOHHOHOOOHOHC26H28O14565mzAPIGENIN-6C-ARAB-8C-GLCC26H28O14565mzOHOHOOH68OOHOHHOOHHOHOOOHHOAPIGENIN-6C-GLC-8C-ARABabcd C.Baiocchietal.Eur.J.MassSpectrom.1911520139intensethanthecorrespondingC-6substitutedonesthisseemstooccurbecauserelativeproductsweremorestable.Neverthelessthesignalintensityothemz427corre-spondingtoa120DalossismuchhigherwhenglucoseisinC-8andthislossunlikethewaterlossesistypicaloglucosemoleculesonly.TheMSMSspectrumoapigeninemonoglycosideglycosylatedatC-8inFigure5bconrmsthisinterpretationshowinganionatmz313whichis120Daromtheprecursorion.HoweversatisactoryresultswerenotalwaysaordedbytheprocessocheckinganalogousregularitiesinallMSMSspectraacquiredinthepositiveionmode.Weattemptedtorationalizemassspectrometricbehaviorodiglycosidederiva-tivesincludingtheinormationthatmaybeinerredrommassdataorthemonoglycosidederivatives.Thiswasduetothepresenceoqualitativelyidenticalspectradieringonlybyaslightdierenceinintensityvalues.ForthisreasonsinceMSMSspectraacquiredinthenegativeionmodeprovideclearerandmorereliableinormationinsomecasestrace-abletosubstitutionpositionsthistypeoexperimentaldatawillchiefybeemployedindiscussingtheseresults.Themolecularidentitiesothediglycosidederivativeswerebyarthemostdiiculttoelucidatebecauseothelargenumberopossiblecombinationscorrespondingtoasingleprecursormzvalue.InadditiontoO-glycosylationandC-glycosylationcombinedO-andC-glycosylationmayalsooccur.Itisalsopossibleortwosugarresiduestoalternativelyoccupytwodistinctsubstitutionpositionsorlinkedtogetherasinglesubstitutionpositioninthelavonoidmolecule.Surprisinglychromatographywasnotomuchhelpinratio-nalizingthisaspectbecauseretentiontimeswerenotalwayssimplyinverselycorrelatedwithincreasingglycosylationtheyweresignicantlyinfuencedbythetypeosugarandtheposi-tionoglycosylation.Inparticulartheeectotheglycosyl-ationpositionappearedtoprevailtotheextentthatinsomecasesdiglycosylatedfavonoidsweremorestronglyretainedthanweremonoglycosylatedones.Table2showsasignicantexampleoretentiontimesodierentglycosidederivativesoapigeninidentiedinplantextracts.Atrstsighttherewasastrikingdierenceinretentiontimesbetween5-Oand7-Oglycosylderivativeswhereasthe8-Cglycosidewasretainedoranintermediatetime.IweassumethatthemolecularmoietiesmostresponsibleorretentionontheC18stationaryphaseareduetothetwobenzeneringsjoinedbytheetherlinkageFigure4andwetrytoestimatetheoverallenhancementopolarityinducedbysiteanddegreeoglycosylationonthebasisoretentiontimeswemayconcludethat5-O-glycosylationsubstantiallychangesthepolarityotheregionotheaglyconemolecule.BeoresubstitutionthisregionischaracterizedbyahydrogenbondbetweentheOHgroupin5andtheneighboringcarbonylgroupwhichmakesthesitelessavailabletopolarinter-actions.OntheotherhandananalogoussubstitutioninPosition7doesnotseemtosignicantlyaltertheretentionothemoleculeduetotheapolarportioneect.Wewillattemptanexplanationorthesephenomenabearinginmindthatthestereochemistryoglycosidebondsisruledbythesp2hybrid-izationothecarbonatomlinkedtooxygenandtheaxialorequatorialpositionotheOHgroupotheglucosemolecule.Theapolarmoietyothemoleculeisnotcompromisedbythe7-Oglycosylationposition.FurtherinspectionothedatainTable2showsthatthepresenceotwosugarscausestheexpectedretentiontimedecreaseversusthemonoglycosylatedderivativeonlywhenthetwosugarsoccupydistinctsubstitutionpositionsitheyarelinkedtogethertheyincreasetheretentiontime.Similarresultsemergeiwecomparethechromatographicbehavioropurestandardswhosestructuraldetailsareexactlyknown.Weveriedthattheorderoelutionoapigeninderivativespurestandardswastheollowing1saponarine2vitexine3vitexine-2-O-rhamnoside4apigenine-7-O-glucoside5apigenine-7-O-neohesperidoside.Thehighnumberoisomericspecieswithanidenticalglycosylationschemehavingquitedierentretentiontimesmakesthechromatographicpatternevenmorepuzzling.Asaconsequencethechromatographicbehaviorothevariousglycosidederivativesdoesnotautomaticallyhelpindeningelutionorder.Usingotherveryinormativerecognitiontech-niquessuchasNMRisnoteasibleinthiscaseduetothelargenumberoconstituentsandtotheslightdierencesintheirretentiontimesmakingthequantitativepuricationprocedureacomplexone.Thusthedeterminationohowthesugarsglucoserhamnoseorarabinosearelinkedtothefavoneapigeninortothefavonolkaemperolmoleculeremainsoprimaryimportancebutisverydicult.SumminguptheexperimentaldatauponwhichtobasetheinvestigationwehavetheUVspectrawhichgiveinorma-tionabouttheaglyconeidentity.SecondasrecommendedbyseveralotherstudieswehaveMSMSinthenegativeionmode.Thisenablesustodistinguish6-Cand8-CglycosylationpositionstoidentiytheaglyconeaswellastodistinguishtheO-glycosylationandmixedCO-glycosylation.AswasstatedaboveMSMSinthepositiveionmodegivescomplemen-taryinormationabout6-Cand8-CglycosylationpositionsbutthatinormationismorediculttointerpretwhereasitprovidesbetterinormationaboutO-glycosylation.ForthesereasonswebasedourdatainterpretationessentiallyonMS2spectraobtainedinthenegativeionmodeandonlyusedMS2spectraobtainedinthepositiveionmodetoexamineO-glycosides.ExhaustiveinterpretationoMSMSspectrawastheonlypossiblewaytoelucidatethemolecularstructureothenumerousconstituentsothisclassocompoundstotheextentpossible.AteranHRMSexploratoryscreeningotheprecursorionspresentinullmassexperimentsitwaspossibletodeter-minealimitednumberoprecursorionmzvaluesuseulorMSMSanalysis.Inthenegativeionmodethesewere609mz593mz579mz577mz563mz447mzand431mz.Mostothesewerecompatiblewithdiglycosylatedderiva-tivesoeitherapigeninorkaemperol.Onlythe609mzand447mzvaluescorrespondtoprobablederivativesokaemp-erol.Consideringthat35constituentswereidentiedinthe 10QualitativeCharacterizationofDesmodiumadscendensConstituentschromatogramofDesmodiumadscendensextractitfollowsthatmanyisomericformsmustbepresent.Figure6illus-tratesthecircumstancesbyshowingasanexamplethreechromatogramsmonitoredinfullmassacquisitionmodeeachonecorrespondingtoisomericprecursorionswiththesameelementalconposition.Tandemmassspectrometryisatleastpartlyabletodiscriminatebetweendifferentisomericspecies.AsanexamplethenegativeionmodeMS2spectraoftwoisomericC-diglycosylatedspeciesatmz563areshowninFigure7theproductionsareformedbycross-ringcleavagesofthesugarfollowingthepatterndescribedinareviewaboutmassspectrometrybehaviorofflavonoids.17Thelossespointedoutinthisreviewareshownindecreasingorderofintensityfollowingthedecreasingstabilityoftheleavingneutralgroup.Indescendingorderthisinvolvesafour-atomoxygenatedcycleathree-atomoxygenatedcycleandafour-carbon-atomcycleasshowninFigure8.ItisknownthatC-glycosylationpositionsnormallyfoundforapigenineandkaempferolareC6andC8.17AsinthiscasetheUVspectraaretypicalofapigenineitfollowsthatthecompoundoriginatingfromtheionat563mzisconsistentwithasymmetricalglycosylationbyglucoseandarabinoseorwithsymmetricalglycosylationbytwomoleculesofrhamnose.AsmaybeseenthetwoMS2spectraarenotqualitativelydifferentforthetwosubstancesbutshowdistinctdifferencesintherela-tiveintensitiesofproductionsignals.ThisdifferenceexcludessymmetricalglycosylationwhichwouldresultinidenticalFigure6.Chromatogramsmonitoredinnegativeionmodebyextractionofthesignalsoftheprecursorions563mza593mzb579mzc.abc C.Baiocchietal.Eur.J.MassSpectrom.19115201311Figure7.MS2spectraoftwodifferentprecursorionsat563mzwithaglyconeAGat269mz.aandbandoftwoisomericionsat593mzwithaglyconesat269mzcandat299mzd.0102030405060708090100RelativeAbundanceA200250300350400450500550600mz563473353503383M-90M-60AG84AG114200250300350400450500550600mz563443473353383M-90M-120AG84AG1140102030405060708090100RelativeAbundanceBM-H-90M-H-120C26H28O14563mzAPIGENIN-6C-GLC-8C-ARABOHOHOOH68OOOHHOOHHOHOOOOHOHOHOOH68OOOHHOOHHOHOOOHOC26H28O14563mzAPIGENIN-6C-GLC-8C-ARABM-H-90M-H-1202030405060708090100RelativeAbundanceC200250300350400450500550600mz593473353503383010AG84AG114M-120M-90200250300350400450500550600mz473383533413503M-90M-120AG84AG114M-60D0102030405060708090100RelativeAbundanceM-H-120M-H-90C27H30O15593mzDIOSMETIN-6C-GLC-8C-ARABOHOHOOH68OOOHOHOHHOHOOOOCH3HOM-H-90M-H-60M-H-120M-H-90C27H30O15593mzAPIGENIN-68C-GLCOHOHOOH68OOOHOHOHHOHOOOHOOHM-H-90M-H-120abcd 12QualitativeCharacterizationofDesmodiumadscendensConstituentsspectra.Suchdierencesarethusimportantandcanbeutilizedorthepurposesointerpretationbytakingintoaccounttheexperimentalevidencethatthe6-C-sugarresidueshowsmoreextensiveragmentationthandoesthe8-C-sugarone.18InthespectrumshowninFigure7athebasepeakcorrespondstoMH90andsignalsoMH60andMH18aremoreabundantthaninspectrumshowninFigure7bdemonstratingthepresenceothepentosearabinoseinPosition6.OnthecontrarythelatterspectrumshowsabasepeakcorrespondingtoMH120typicalragmentation02XoahexosewhichmustthereorebeinPosition6.Twootherimportantionscanbeobservedinthetwospectraduetotheaglyconeplustheragmentsothesugarsthatremainlinkedtoitatercross-ringcleavagesi.e.A84andA114.Thefrstioncorrespondstotheaglyconeplustheragmentremainingaterthelosso120Daand90Dabotha02Xlossromthehexoseandpentosesugarsrespec-tivelythesecondioncorrespondstothemixedlosses02Xohexoseand03Xopentoseorconversely03Xohexoseand02Xopentose.TheseionsenableustodeterminetheaglyconeprovidingcomplementaryinormationtotheUV-spectrumisuchaspectrumislackingthenitbecomesessentialtodeterminetheaglycone.TheMS2spectrainthecaseotwoisomersatmz593isreportedinFigures7candd.Inthespectrumo7cA84andA114ionsarerespec-tivelyatmz353andmz383correspondingtotheapigenineaglyconesmz269.InthespectrumshowninFigure7dthesametwoionslieatmz383andmz413respectivelycorrespondingtoaMHionoanaglyconeat299mz.ThislatternegativeiondoesnotcorrespondtotectorigenineacomponentreportedtobepresentinDesmodiumadscendensbecausethislattercompoundhasamethoxygroupinC6whereasthetwospectrainFigures7canddaretypicaloanasymmetricalglycosylationinC6andC8.MorelikelyitcouldbeC-diglycosylateddiosmetinwhichisamethoxylatedapigeninebutwiththemethoxygroupinPosition4otheaglyconeringB.Figures9aandbshowtwoexamplesoMS2spectraobtainedinthepositiveionmoderelativetoO-diglycosylatedderivativesrespectivelyoapigenineFigure9aandokaemperolFigure9b.Thespectraareverysimpleanditisstraightorwardtodeducethekindosugarandtheaglycone.Thetwosugarsaresurelylinkedtogetherbya1-6interglycosidicbondbecausethelossotheexternalsugargivesasignalhigherthanthatothelossotheinternalone.12Howeverthereisnoindicationotheglycosylationpositionsotheattributioncanonlybemadeonthebasisoretentiontimes.ForthisitmustbetakenintoaccountthattheO-glycosylationin5islesscommonbecausetheOHinthispositionparticipatesinahydrogenbondwiththeoxygenothecarbonylandalsothat7-glycosylationinduceslongerretentiontimesthandoesglycosylationatanyotherposition.1920ThislatterpointisurthersupportedbytheexperimentalchromatographicrunsoaseriesodierentC-andO-mono-anddi-glycosidesoapigenineandkaemperolseeTable2.Figure9cshowsaMS2massspectruminthepositiveionmodeoanO-di-glycosylderivativeoanaglyconemoleculeomzvalue301.BesidesthisMHmzvaluethereisnourthersupporttoconfrmidentifcationotheaglyconeasdiosmetin.Howeverthecontemporarypresenceintheplantodi-C-glycosylderivativesodiosmetinleadstothereason-ableconclusionthattheO-glycosylatedderivativeisalsoothesamespecies.Figure9dshowstheMS2spectruminthenegativeionmodeothe7-O-rhamnosylquercetinthisisthesoleexampleOHOHOHOHC5H10O4134.057DaPENTOSELEAVINGGROUPOOHOHC3H6O390.031Da15X03X02XOOHC2H4O260.021DaOHOHOHC4H8O3104.047DaOOHOHOOHOHOHC4H8O4120.042DaC3H6O390.031DaLEAVINGGROUPHEXOSE02X03X15XFigure8.Typicalsugarfragmentationneutrallossesinvolvingcross-ringcleavagesdetectedbyHRMS2. C.Baiocchietal.Eur.J.MassSpectrom.19115201313Figure9.MS2spectraoprecursorionsat563mza581mzb595mzcand447mzdidentifedasaglycosylatedquercetine.200250300350400450500550600mz5474332710102030405060708090100RelativeAbundanceM-132M-132-162M-162M-132APIGENIN-7O-DIGLCC26H29O14565mzOOHHOHOOHOHOOHO7OOHOHOOH200250300350400450500550600mz449563287AB0102030405060708090100RelativeAbundanceM-132-162M-132M-162M-132OOHHOHOOHOHOOHO7OOHOHOOHOHC26H29O15581mzKAEMPFEROL-7O-DIGLC140180220260300340380420460500mz301447200250300350400450500550600mz4633010102030405060708090100RelativeAbundanceM-132M-132-162CD0102030405060708090100RelativeAbundanceM-146C27H30O15595mzDIOSMETIN-7O-DIGLCOOHHOHOOHOHOOHO7OOHOHOOHOCH3M-132M-162M-1467-O-RHAMNOSYL-QUERCETINC21H20O11447mzHOHOOHOCH3O7OOOHOOHOHabcd 14QualitativeCharacterizationofDesmodiumadscendensConstituentsoanaglyconeotherthanapigeninorkaemperoloundintheplantextract.AnexaminationoalltheUVandmassspectrometricdatatogetherwithsuitableinterpretationleadtothelistomainconstituentsoDesmodiumadscendensinthefavonoidclassreportedinTable2.ConclusionsDesmodiumadscendensisaplantexhibitinginterestingposi-tiveeectsagainstmanydiseasesthatischiefyusedinArica.ItsongoingintroductionontotheEuropeanherbalextractmarketmeansthereisaneedorreliablequalitycontrolanal-ysis.Atpresentnocomprehensiveanalysismethodoitsconstituentsexists.Thisstudyattemptedtodetermineaprotocoloqualitativeanalysisothedierentclassesocompoundspresentintheaerialpartotheplant.Thethreemostsignicantclassesocompoundsknowntobepresentinthisplantexhibitveryvariedchemicalpropertiesandthusrequiredierentextrac-tionmodesalkaloidsversussoyasaponinsandfavonoidsbutabovealltheyneeddierentchromatographicsepara-tionanddetectionconditions.HRMSwasaveryuseuldetec-tionsysteminthecaseosaponinsandalkaloidsbutwasomodestuseulnessinthecaseofavonoids.Thislatterclasscontainsalargenumberocompoundsinmostowhichalimitednumberoaglyconesarepresentinvariousglycosylatedormsotengivingrisetoisomericspeciesthatareindistinguishableeveninhigh-resolutionmassspectra.InthiscasediodearrayUVdatacombinedwithcareulinterpretationoMSMSdatainbothpositiveandnegativeionmodescanbeutilizedtoidentiysomeconstituents.Howeversomequestionsremainopenaboutthecasesinwhichthenumberodetectedisomerswassuperiortothedierentpossiblecombinationsoaspecicaglyconewithsugarglyco-sylationpositions.Thiswasorexamplethecaseomzvalues563andmz593whichexhibitedourasymmetrictwobytwoidenticalC-diglycosidesinsteadotwowithourdistinctretentiontimes.Apossibleexplanationmaybethatdierentisomersohexoseorpentosewithdiversestereo-chemistryotheOHgroupsmayparticipateintheirstructureorexamplemannoseinsteadoglucoseorxylitolinsteadoarabinose.Bethisasitmaythelistresultingromouranalysisisarricherthanotherspublishedpreviouslyandshowsthatfavonoidscompriseaconspicuousproportionothecompoundscharacterizingtheplant.Hithertoneglectedthesemayinutureplayanimportantroleinstudyingtheplantsproperties.References1.O.AmpooPlantsthathealWorldHealth261391977.2.O.B.McManusG.H.HarrysK.M.GiangiacomoP.FeigenbaumJ.P.ReubenM.E.AddyJ.F.BurkaG.J.KaczorowskyandM.L.GarciaAnactivatorocalcium-dependentpotassiumchannelsisolatedromamedici-nalherbBiochemistry3261281993.doi10.1021bi00075a0023.M.E.AddyandE.M.K.AwumeyEectsotheextractsoDesmodiumadscendensonanaphylaxisJ.Ethnopharmacol.112831984.doi10.10160378-87418490074-64.M.E.AddyandW.K.DzanduDose-responseeectsoDesmodiumadscendensaqueousextractonhistamineresponsecontentandanaphylacticreactionsintheguineapigJ.Ethnopharmacol.18131986.doi10.10160378-87418690039-55.M.E.AddyPharmacologicalactionsoDesmodiumadscendensaGhanaianmedicinalplantDiscov.Innov.52051993.6.M.E.AddySomesecondaryplantmetabolitesinDesmodiumadscendensandtheireectsonarachidonicacidmetabolismProstag.Leukotr.Ess.47851992.doi10.10160952-32789290191-K7.O.N.OgbeideandM.ParvezIdenticationothefavonoidsinpapilionaceaefowersusingpaperchromatographyJ.LiquidChromatogr.1529891992.doi10.1080108260792080163658.S.Asante-PokuJ.SakakibaraandM.E.AddyAlkaloidsoDesmodiumadscendensGhanaMed.J.221111988.9.M.E.AddySeveralchromatographicallydistinctractionsoDesmodiumadscendensinhibitsmoothmusclecontractionsInt.J.CrudeDrugRes.2811989.10.P.NgouemoM.Baldy-MoulinierandC.Nguemby-BinaEectsoanethanolicextractoDesmodiumadscendensoncentralnervoussysteminrodentsJ.Ethnopharmacol.52771996.doi10.10160378-87419601389-X11.G.S.BarretoEectobutanolicractionoDesmodiumadscendensontheanococcygeusotheratBrazilJBiol.622232002.doi10.1590S1519-6984200200020000512.J.PothierJ.RagotandN.GalandPlanarchromatographicstudyofavonoidsandsoyasaponinsorvalidationongerprintsoDesmodiumadscendensodierentoriginJ.Plan.Chromatogr.191912006.doi10.1556JPC.19.2006.3.313.A.G.MarshallandC.L.HendricksonHigh-resolutionmassspectrometersAnnu.Rev.Anal.Chem.15792008.doi10.1146annurev.anchem.1.031207.11294514.M.ShigelovaandA.MakarovAdvancesinbioanalyticalLC-MSusingtheOrbitrapmassanalyzer2Bioanalysis17412009.doi10.4155bio.09.6515.A.MakarovandM.ShigelovaCouplingliquidchromatographytoOrbitrapmassspectrometryJ.Chromatogr.A121739382010.doi10.1016j.chroma.2010.02.02216.Y.L.MaF.CuyckensH.VandeHeuvelandM.ClaeysMassspectrometricmethodsorthecharacterisation C.Baiocchietal.Eur.J.MassSpectrom.19115201315anddierentiationoisomericO-diglycosylfavonoidsPhytochem.Anal.121592001.doi10.1002pca.57317.F.CuyckensandM.ClaeysMassspectrometryinthestructuralanalysisofavonoidsJ.MassSpectrom.3912004.doi10.1002jms.58518.F.FerreresB.M.SilvaP.B.AndradeR.M.SeabraandM.A.FerreiraApproachtothestudyoC-glycosylfavonesbyiontrapHPLC-PAD-ESIMSMSApplicationtoseedsoquinceCydoniaoblongaPhytochem.Anal.143522003.doi10.1002pca.72719.G.C.KiteE.A.PorterF.C.DenisonR.J.GrayerN.C.VeitchI.ButlerandM.S.J.SimmondsData-directedscansequenceorthegen-eralassignmentoC-glycosylfavoneO-glycosydesinplantextractsbyliquidchromatography-iontrapmassspectrometryJ.Chromatogr.A11041232006.doi10.1016j.chroma.2005.11.07020.R.J.GrayerG.C.KiteM.Abou-zaidandL.J.ArcherTheapplicationoatmosphericpressurechemicalionizationliquidchromatography-massspectrometryintheche-motaxonomicstudyofavonoidscharacterisationofavonoidsromOcimumgratissimumvar.gratissimumPhytochem.Anal.112572000.doi10.10021099-1565200007081143.0.CO2-A 17ISSN1469-0667IMPublicationsLLP2013doi10.1255ejms.1212AllrightsreservedEUROPEANJOURNALOFMASSSPECTROMETRYSincetyrosineresidueswereirstlinkedtomalignancy1anunderstandingothelinkbetweencancerandcellularsignallinghasbeenestablished.24Asaresultoverthelastdecadethepharmaceuticalindustryhasproducedanumberooncogenicsignalblockers.56The2012FDAapproveddrugslistcontains14tyrosinekinaseinhibitorsincludingtensmallmoleculesandourmonoclonalantibodieswithotherspending.Predictingpatientresponsetosuchanti-cancerdrugsisessentialorthesuccessothetreatmentanditscosteectiveness.Detectionotheepidermalgrowthactorreceptoramphiregulinandepiregulininormalin-ixedparain-embeddedhumanplacentatissuebymatrix-assistedlaserdesorptionionizationmassspectrometryimagingKhaledMahmoudadLauraM.ColebJillianNewtonbSabahMohamedcMaherAl-EnaziaPhilQuirkedandMalcolmRClenchbaSchooloMedicineAl-JouUniversitySakakaKingdomoSaudiArabiabBiomedicalResearchCentreShefeldHallamUniversityS11WBUKcFacultyoSciencesAinShamsUniversityCairoEgyptdLeedsInstituteoMolecularMedicineUniversityoLeedsLS29JTUK.E-mailk.mahmoudleeds.ac.ukThestudyotheexpressionandthetissuedistributionothetyrosinekinasedrug-targetepidermalgrowthactorreceptorEGFRisointerestinoncologyasamarkeropotentialefcacyotreatment.Ithasbeenreportedhoweverthattheresponseratestoanti-EGFRdrugsarepoorlylinkedtoitsexpression.ClinicalstudieshavealsorevealedapatientresponsecorrelationwiththeexpressionlevelsotwoEGFRligandsamphiregulinandepiregulin.Herewereportthedevelopmentoamatrix-assistedlaserdesorptionionisationmassspectrometryimagingmethodologyorthestudyoEGFRepiregulinandamphiregulindistributioninormalinfxedparafnembed-dedhumanplacentaltissueandacomparisontoexpressionpatternsobtainedbyimmunohiostochemistry.Usingon-tissuedigestsandimagingospecifcpeptidesthetissuedistributionotheseproteinshasbeenobtaineddownto30mspatialresolution.KeywordsMALDItissueimagingrecombinantcancertherapeuticsEGFRamphiregulinepiregulinFFPEplacentaCetuximabPanitumumabimmunohistochemistrycolorectalcancertargeteddrugsIntroductionK.Mahmoudetal.Eur.J.MassSpectrom.1917282013Received8November2012nRevised27February2013nAccepted27February2013nPublication4March2013 18DetectionofEGFREpiregulinandAmphiregulininFFPEHumanPlacentalTissueClinicalstudieshavedemonstratedacorrelationbetweenpatientresponsetotyrosinekinaseblockingdrugsandarangeointrinsicactors.Theseincludethegenestructureandexpressionlevelaswellastheactivationstatusothetargettyrosinekinase.714Thedevelopmentoacquiredresist-anceurthercomplicatesthesituation.1521Incolorectalcancerithasbeenoundthatpatientresponseratestoanti-epidermalgrowthactorreceptorEGFRdrugsarepoorlylinkedtoEGFRexpression.2224Objectiveresponseswereobservedinpatientswithnega-tiveandlowexpressionlevels.25ThesediscrepanciesweresuggestedtoariseasaresultopoorEGFRassaysortorelatetoapoorunderstandingoitsunction.2627Despitetheapprovalosomeprognosticgenetictests2833suchcontro-versialresultsarestillanissue.3436OtherstudieshavelinkedpatientresponsewiththeexpressionlevelsothetwoEGFRligandsamphiregulinandepiregulin.3742Addressingthelimitationsothecurrentlyusedassayswouldbeoclearpatientbeneft.43Thisisparticularlyimportantincaseswheretheuseoanti-EGFRtherapeuticsrequiresareliablecriterionorpatientselection.Matrix-assistedlaserdesorptionionisation-massspec-trometryimagingMALDI-MSIisanadvancedanalyt-icaltoolthatallowsmolecularproilingandimagingoseveralclassesobiologicalcompoundsdirectlywithintissuesections.Usinginsitutrypticdigestionasasamplepre-treatmentpriortoMALDI-MSIanalysisenablesthelocalisationandtheidentiicationoproteinsdirectlyromarchivedormalin-ixedparain-embeddedFFPEsamples.44Thesearethestandardtypeopreservedtissuesinclinicalpractice.Theabilitytostudythedistributionandtoidentiyproteinbiomarkersdirectlyromarchivedtumoursampleswithknownoutcomeisoimportantclinicalinterest.WehavepreviouslydemonstratedthatMALDI-MSIoFFPEtumoursectionscouldbeusedtostudythedistributionothestressproteinGRP7845andthatasystemortheclassiicationopancreaticcancertumourscouldbegeneratedromtheMALDI-MSIstudyoapancreaticcancerFFPEtissuemicroarrayTMA.46InthestudyoEGFRJiang47hasreportedaMALDIproce-dureorEGFRidentiicationinextractsobreastcancercelllinesandTaguchi48andRoder49haveusedMSserumanalysisorpredictingresponsetothesmallmoleculetyrosinedruginhibitorsIressaandTarseva.HoweverthedirectMALDI-MSimagingoEGFRoritstwoligandsamphiregulinandepiregulininhumantissueshasnotbeenreported.IntheworkdescribedhereMALDI-MSIhasbeenusedorthefrsttimetostudythedistributionoEGFRanditstwoligandsamphiregulinandepiregulininFFPEtissue.Humanplacentaltissuehasbeenusedasamodelsystemorthisinitialworkowingtothehighabundanceotheseproteinsinthistissuetype.Thedevelopmentothismethod-ologycomplementsthecurrentarsenaloassaysandopensopportunitiesorhighthroughputanalysisothelargeFFPEarchivalcollectionsorclinicaldiagnosis.MaterialsandmethodsTissuesConsentedanonymoushumanplacentawasobtainedwithethicalapprovaloritsuse.PositivecontrolsRecombinantEGFRamphiregulinandepiregulinwerepurchasedromStratechUK.TissuefxationTissuesampleswerefxedin10bueredormalinor24hdehydratedin70EtOHandparafnembedded.5msectionswerecutusingamicrotomeLeicaMicrosystemsUKandmountedontoahistologicalglassslide.FFPEtissuesectionswerestoredatroomtemperatureuntilurtheranalysis.TissuepreparationParafnwasremovedromFFPEtissuesectionsbyimmersingthesampletwiceinxylenesolutionor10mineachtimeinordertocompletelyremovetheembeddingparain.Thesectionsweregentlyhydratedor3minpersolutionin100EtOH95EtOHand70EtOHconsecutively.Antigenretrievalwasperormedbyheatinginamicrowaveovenor4minat50powerinatri-sodiumcitratebuerat0.01MpH6.3.Thesectionwascooledtoroomtemperaturerinsedwithwaterandthenallowedtodry.Itwasthenwashedor1mineachin70and90EtOHbeorebeingimmersedinchloroormor15s.ItwasthenallowedtoairdrypriortotrypsinandmatrixdepositionTheantigenretrievalstepwascrucialorthesuccessuldetectionopeptidesromthesesamples.In-solutionandin-situdigestionAlltrypsinusedinthisstudywaspreparedat20gmL1in50mMammoniumbicarbonatebuerpH8.4containing0.5Octyl-ab-glucosideOcGlc.Therecombinantstandardsweredigestedinsolutionusingtheollowingmethod10Lorecombinantprotein10gwasreducedandalkylated.OneLotrypsinsolutionwasthenaddedandthediges-tionperormedor3hinashakingincubatorat37Cinahumidenvironment.Aurther1Lotrypsinwasthenaddedtothetubewhichwasurtherincubatedovernightintheshakingincubatorat30C.ThedigestionwasstoppedwithiceandtheresultingpeptidesolutionpurifedusingZipTipC18pipettetipsMilliporeHampshireUKbeoreMALDI-IMS-MSIanalysis.In-situdigestionwasperormedonFFPEplacentaltissuesectionsunderhumidconditionsthetrypsinsolutionwassprayedontothesectionsinaseriesofvelayersatalowrate2Lmin1usingaSunCollectautomaticsprayerSunChromGermany.Sectionswerethenincubatedover-nightat37C5CO2.MatrixdepositionFivemgmL1a-CHCAmixedwithanilineANIwassprayedontothesectionusingtheSunCollectautomaticsprayer. K.Mahmoudetal.Eur.J.MassSpectrom.191728201319AnilinewasaddedtotheCHCAsolutioninequimolaramountstotheCHCAaddedi.e.5mgmL1CHCAmatrixsolutionwillcontain2.4Laniline.Thefrstandsecondlayersweresprayedat3Lmin1toallowamatrixseedingprocess.Threesubsequentlayersweresprayedat3.5Lmin1.MassspectrometryandimagingmassspectrometryPeptidemassingerprintsandimageswereacquiredbyMALDI-MSMSIusingeitheraQ-StarPulsar-ihybridquadrupoletime-o-lightmassspectrometerAppliedBiosystemsMDSSciexOntarioCanadaorSYNAPTG2HDMSsystemWatersCorporationManchesterUK.InthecaseotheQ-StardatawasacquiredusingoMALDIServer5.1AppliedBiosystemsMDSSciexOntarioCanadaasAnalystQS1.1AppliedBiosystemsMDSSciexOntarioCanada.wiiles.TheseweresubsequentlyconvertedintoAnalyze7.5fleormatbytheoMALDIserversotwareorimagegenera-tionusingthereelyavailableBioMap3.7.5.5sotwarewww.maldi-msi.org.InthecaseotheSynaptG2HDMSinstrumentdatawasacquiredusingMassLynxWatersCorporationManchesterUKandconvertedtoAnalyze7.5fleormatorvisualisationinBioMapusingtheWatersMALDI-Imagingconvertersotware.DataproducedwithionmobilityseparationwasproducedusingWatersHDIImagingsotwareusingtheollowingparameterssotwareversionHDI1.1WatersCorporationUKspecifcitytypeIMSMSNumberomostintensepeaks1000resolu-tion10000lowenergyintensitythreshold50.Thelowintensitythresholdwastoallowlowabundantspeciestobeincluded.BothotheseinstrumentsarefttedwithavariablerepetitionrateNdYAGlaserwhichwassetto1kHz.Initialimageacqui-sitionwasperormedusingraster-imagingmodeat150mspatialresolutionontheQ-Starinstrumentandsubsequent30mimageswereacquiredontheSynaptwithionmobilityseparation.Toenablesimplevisualcomparisonbetweenimagesalldatawasnormalisedtoeithermz877ormz1066signalsarisingromtheaCHCAmatrix.Inordertoevaluatethemassmeasurementerrorinthe150mimagingdatasetanaveragemassspectrumwascreatedromtheentireimageflecollectedat150mspatialresolutionontheAppliedBiosystemsQ-Starinstrument.Thiswasdonebyaveragingtheentirechromatographicrepre-sentationothedatasetusingtheAppliedBiosystemAnalystQS1.1sotware.TheinstrumentwascalibratedpriortotheanalysisbeingperormedwithPEG1000.ImmunohistochemistryTissuesweresectionedat5mcollectedonPlusFrostslidesSolmediaLtdUKdriedovernightat37Candthenheatedat70Cor20min.SlidesweredewaxedantigenretrievedinAccessrevelationsolutionA.MenariniDiagnosticsLtdUKheatedto125Cor2mincooledto90CwashedinslidewashbuerA.MenariniDiagnosticsLtdUKat90CthenstainedusinganX-CellpluskitA.MenariniDiagnosticsLtdUKTheollowingprimaryantibodieswereusedmonoclonalmouseanti-EGFRantibodycloneEGFR25LeicaUKdilution175rabbitpolyclonalanti-amphiregulinantibodyAbgentUSAdilution140andrabbitpolyclonalanti-epiregulinanti-bodyAbgentUSAdilution150.SerialsectionswereusedorMALDI-MSIandimmunohistochemistry.ResultsanddiscussionThehumanplacentaiseasilyaccessibleorstudyandisknowntoproducemanygrowthactorsreceptorsenzymesandhormones.5052TheexpressionoEGFRandtheepidermalgrowthactorEGFhasbeenpreviouslyinvestigatedinthehumanplacentabyimmunohistochemistry.5355OurdataFigure1aareinagreementwiththesereports.Stainingshowedahighmembranouslocalizationinthesyncytiotrophoblastthevillousandextravillouscytotrophoblastaswellasthedecidualcells.TheamphiregulinimmunohistochemistryresultsFigure1barealsoinagreementwithapreviousreport56andwiththeexpressionpatternseenorthisproteinandEGFRintheHumanProteinAtlas.AlthoughtheimmunohistochemistrytissuedistributionoepiregulinwasnotdescribedinthehumanplacentaandisnotavailableintheHumanProteinAtlashighexpressionothetranscriptwasreported.57OurresultsindicatethattheepiregulindistributioninthehumanplacentahasasimilarpatterntoamphiregulinalthoughtheintensityishigherFigure1c.TheMALDIpeptidemassfngerprintgeneratedromthein-solutiondigestothepositivecontrolrecombinantEGFRisshowninFigure2aclearlyvisibleistheEGFRtrypticpeptide963MHLPSPTDSNFYR975atmz1564.7.ThishaspreviouslybeendemonstratedtogivehighsensitivityinMALDI-MS47andisalsoobservablewithgoodintensityintheworkreportedhere.Table1showstheaccuratemassesobtainedromtheaveragedmassspectrumacquiredacrossthetissuesampleorthepeptidesstudiedthesedataareromthe150mimagerecordedontheQStarinstrument.Theresultsrangerom3ppmto46ppmwithanaverageo15pmmandareinkeepingwiththespecifcationotheinstrument.ThedistributionoEGFRepiregulinandamphiregulinin5mFFPEhumanplacentasectionsstudiedbyMALDI-MSIat150mspatialresolutionareshowninFigures2be.Figure2bshowstheMALDI-MSIimageoEGFRdistribu-tiongeneratedusingthedistributionotheon-tissuedigestproducedtrypticpeptide963MHLPSPTDSNFYR975theoreticalmz1564.721.Therelativeintensityanddistributionothispeptideinthe150mMALDI-MSIimageisingoodagree-mentwiththedatashowninthelowmagniicationIHCimageFigure1.AlsovisibleistheconfrmatorysignalintherecombinantEGFRpositivecontrolspotarrow.Figure2cshowstheMALDI-MSIimageothedistributionoepiregulinusingthetrypticpeptide93CEVGYTGVR102theoreticalmz983.462.Thesignalromthepositivecontrolepiregulinisalsoclearlyobservablearrow.Plates2dand2eshowMALDI-MSIimagesothedistributionoamphiregulinusingthetrypticpeptides172CQQEYFGER180theoreticalmz 20DetectionoEGFREpiregulinandAmphiregulininFFPEHumanPlacentalTissue1159.484and104VEQVVKPPQNK114theoreticalmz1265.721respectively.Thecorrespondingsignalsinthecontrolrecom-binantamphiregulinarealsoclearlyvisiblearrows.Thetwoimagesshowverymuchthesamedistributionalthoughtheyappearslightlydierentonfrstinspectionowingtothedierentintensitiesothesetwoions.Figure1.PatternoexpressionoaEGFreceptorbamphiregulinandcepiregulinbyimmunohistochemistryperormeduponHumanplacenta.Theupperrightinsetwindowisothewholeslidewiththemagnifedareaoutlinedbythesmallredbox.Thecorre-spondingMALDIimagesospecifcpeptidesortheseproteinsareshownorreerenceinthebottomletotheimages. K.Mahmoudetal.Eur.J.MassSpectrom.191728201321Figure2.MALDIfngerprintandMALDI-MSIin5mFFPEhumanplacentasectionsat150mmresolutionaMALDIpeptidemassfngerprintgeneratedromthein-solutiondigestothepositivecontrolrecombinantEGFR.ClearlyvisibleistheEGFRtrypticpeptide963MHLPSPTDSNFYR975atmz1564.721.bMALDI-MSIimageothedistributionandtherelativeintensityotheEGFRpeptide963MHLPSPTDSNFYR975atmz1564.721.AlsovisibleistheconfrmatorysignalintherecombinantEGFRpositivecontrolspotarrow.cMALDI-MSIimageothedistributionoepiregulinEREGusingthetrypticpeptide93CEVGYTGVR102mz983.462.Thesignalromthepositivecontrolepiregulinisalsoclearlyobservablearrow.dandeMALDI-MSIimagesothedistributionoamphiregulinAREGusingthetrypticpeptides172CQQEYFGER180mz1159.484and104VEQVVKPPQNK114mz1265.721respectively.Thecorrespondingsignalsromthecontrolrecombinantamphiregulinarealsoclearlyvisiblearrows. 22DetectionofEGFREpiregulinandAmphiregulininFFPEHumanPlacentalTissueOneareaoconcerninMALDI-MSIwhenon-tissuedigestsareperormedisthedelocalisationoproteinscausedbythelargenumberostepsinsamplepreparationincludingsprayingoenzymesandmatrices.Thisdoesnotappeartobeanissuewiththeprotocolreportedhere.Figures3bhshowthedistributionsoactinEGFRandepiregulinsignalsin5mFFPEhumanplacentasectionrecordedbyMALDI-MSIat30mspatialresolution.Figure3ashowsanoverviewotheplacentasectionpriortoMALDI-MSIanalysiswiththeregionshadedinredhavingbeenimagedbyMALDI-MSIat30mresolution.ThelocationsotherecombinantEGFRepiregulinandamphiregulincontrolsareshownasthreedarkdotsatthetoprightcornerotheborderedregion.Figure3bshowstheregistrationotheshadedareaonadigitalscanoahumanplacenta.Figure3cshowstheMALDI-MSIimageothedistributionotheCHCAMALDImatrixpeakatmz1066usedornormalizationandinverseco-registrationinthemarkedarea.Figure3dshowsaMALDI-MSIimageothedistributionoactinatmz1198.7usedtoindicatethevalidityothestandards.Theboxedregionismagnifedonthelet.Plates3eandshowMALDI-MSIimagesoEGFRusingtrypticpeptides978YLVIQGDER986theoreticalmz1092.568and963MHLPSPTDSNFYR975theoreticalmz1564.72theboxedregionsaremagnifedonthelet.Plates3gandhshowMALDI-MSIimagesoepiregulinusingtrypticpeptides93CEVGYTGVR102theoreticalmz983.462and151EPKKEYER158theoreticalmz1078.553respectivelytheboxedregionsaremagnifedonthelet.InMALDI-MSIanumberomethodologieshavebeenreportedorconfrmationopeptideidentitywhenon-tissuedigestsareusedtheseincludetheuseoionmobilitysepara-tioncoupledtoMSMS4557accuratemass58andtheuseoposi-tivecontrols.45InthedatareportedherethesignalsrecordedorthepeptidesstudiedwerenotosuicientintensitytoallowMSMSspectratoberecordeddirectlyromthetissue.Hencetheconfrmationoassignmentisbasedonmatchingthemzvalueandionmobilitydrittimeorthestudiedpeakwiththoseothecorrespondingpeptidegeneratedromthepositivecontrol.Figures4acdemonstratehowthisisachievedusingWatersHDIImagingsotware.Figure4ashowstheMALDI-MSIimageothedistributionEGFRtrypticpeptide963MHLPSPTDSNFYR975theoreticalmz1564.721.ThemzorimaginginthesedatawasselectedbyclickingontheappropriatepeakintherepresentationotheionmobilityseparationotheionsgeneratedromthespotothedigestothepositivecontrolFigure4c.AnexpansionothemassspectrumothepositivecontroldigestisshownasFigure4bandorcomparisonaregionotheon-tissuedigestisshownasFigure4d.AscanbeseentheEGFRpeptideindi-catedwithanasteriskisoverylowabundanceinthesedataanditwouldbeproblematictoconfdentlyassignitsidentityiitdidnotalsomatchinbothaccuratemassandionmobilitydrittimetothecorrespondingioninthepositivecontrol.Thelackouniversallyacceptedstandardisationguidelinesgoverningimmunohistochemistrymakesthedataobtainedhighlydependentuponthetypeoantibodytheprotocolsthescoringsystemandthecut-osused.59Thispaperdemon-stratestheabilitytovisualisethepresenceopotentiallyclinicallyimportantproteinsinhumanFFPEtissuesusinganalternativemethodtoIHCi.eMALDI-MSI.TheadvantagesothistechniqueincludenorequirementoranantibodythatworksinFFPEandthatitisnotdependantonexpo-sureotheantigentotheantibodyortheconormationotheprotein.Inadditionthepeptidesequencesusedoridentifca-tioncanbechosenbytheinvestigatorandcorroboratedusingotheruniquepeptidesequenceswithinthesamemolecule.Multipleproteinscanalsobesimultaneouslyvisualisedonthesameslide.CurrentlyourknowledgeotheimportanceoamphiregulinandepiregulinexpressioninKRASandBRAFwildtypecancersisinsufcienttodeterminewhethertheyiden-tiyauniquelysensitivepopulationopatientswithcolorectalcancerwhowillrespondtoCetuximaborPanitumumabtreat-ment.AlsodespitetheassociationoEGFRoverexpressionwithaggressiveandpoordiagnosisometastaticcolorectalcancer60itwaspoorlylinkedtopatientresponsetoanti-EGFRdrugs.2224ThesedrugssuchasthemonoclonalantibodiesCetuximanandPanitumumabshowedasignifcantefciencyintreatingCRCpatientsandwerelinkedtoimprovingtheoverallsurvivalothemetastaticCRCpatients.61HoweverurtherstudiesusingMALDI-MSIonclinicaltrialpopulationstreatedwiththesedrugsmayanswerthequestions.InutureworkthemethodologyreportedherewillbeappliedtoFFPEProteinPeptidesequenceTheoreticalmass3d.p.Experimentalmass3d.p.ErrorEGFRMHLPSPTDSNFYRYLVIQGDERIICAQQCSGREYHAEGGK1564.7211092.5681078.513890.4001564.7241092.5921078.528890.4312ppm22ppm14ppm34ppmEpiregulinEPKKEYERKEYER1078.553724.3621078.560724.3607ppm3ppmAmphiregulinVEQVVKPPQNKCQQEYFGER1265.7211159.4841265.7801159.47546ppm8ppmTable1. K.Mahmoudetal.Eur.J.MassSpectrom.191728201323Figure3.ThedistributionoEGFRepiregulinandamphiregulinsignalsin5mFFPEhumanplacentasectionat30mresolution.aOverviewotheplacentasectionpriortoMALDI-MSIanalysis.TheregionshadedinredwasimagedbyMALDI-MSIat30mresolution.bApproximateregistrationotheshadedareaonadigitalscanoaplacentasection.ThelocationsotherecombinantEGFRepiregulinandamphiregulincontrolsinsequenceromlettorightareshownasthreedarkdotscMALDI-MSIdistributionotheCHCAMALDImatrixpeakatmz1066.dMALDI-MSIdistributionoactinatmz1198.7.Theborderedregionisenlargedonthelet.eMALDI-MSIimagesoEGFRusingtrypticpeptides978YLVIQGDER986mz1092.568.Theborderedregionisenlargedonthelet.MALDI-MSIimagesoEGFRusingtrypticpeptides963MHLPSPTDSNFYR975mz1564.72.Theborderedregionisenlargedonthelet.gMALDI-MSIimagesoepiregulinusingtrypticpeptides93CEVGYTGVR102mz983.462.Theborderedregionisenlargedonthelet.hMALDI-MSIimagesoepiregulinusingtrypticpeptides151EPKKEYER158mz1078.553.Theborderedregionisenlargedonthelet.Acolourversionothisfgureisavailableintheonlineversion. 24DetectionoEGFREpiregulinandAmphiregulininFFPEHumanPlacentalTissuedFigure4.IsolationoPeptideSignaloInterestusingWatersHDIImagingsotware.aMALDI-MSIimageothedistributionEGFRtrypticpeptide963MHLPSPTDSNFYR975theoreticalmz1564.721romaFFPEplacentasample.bExpandedregionothepositivecontroldigestpeptidemassfngerprintaregioncrepresentationotheionmobilityseparationotheionsgeneratedromthespotothedigestothepositivecontrolwiththeionointerestshowninreddexpandedregionoaMALDImassspectrumtakendirectlyromtheon-tissuedigest.AscanbeseentheEGFRpeptideindicatedwithanasterixisoverylowabundanceintheon-tissuedigestdataanditwouldbeproblematictoconfdentlyassignitsidentityiitdidnotalsomatchinbothaccuratemassandionmobilitydrittimetothecorrespondingioninthepositivecontrol.Acolourversionothisfgureisavailableintheonlineversion. 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29ISSN1469-0667IMPublicationsLLP2013doi10.1255ejms.1215AllrightsreservedEUROPEANJOURNALOFMASSSPECTROMETRYMatrix-assistedlaserdesorptionionizationtime-o-lightmassspectrometryMALDI-ToFMSiswidelyusedortheanalysisobiomolecules.ThechoiceomatrixisbasedonanempiricalruleinMALDI-MS.AttheearlystageoMALDIexperimentstwotechniqueswerereported.Oneisthatfnecobaltparticlesmixedwithglycerolwereusedorionizationoproteins.1Theotheristheuseonicotinicacidasamatrix.2Thesetechniqueshavebeenimprovedasaliquidmatrixandasolidmatrixmethodrespectively.InasolidmatrixcinnamicacidderivativeshavebeenrecognizedasgoodMALDImatricesorproteins.3Thesematriceshaveseveraladvantagesoverthenicotinicacid.Thematrix25-dihydroxybenzoicacid25-DHBwasalsoreportedasasimilarorbettermatrixthanthecinnamicacidderivatives.4RecentlyinproteomeandAthiophene-containingcompoundasamatrixormatrix-assistedlaserdesorptionionizationmassspectrometryandtheelectricalconductivityomatrixcrystalsAkikazuYasudaaTakayukiIshimaruaShogoNishiharabMasamichiSakaiaHideyaKawasakibRyuichiArakawabandYasushiShigeriaaHealthResearchInstituteNationalInstituteoAdvancedIndustrialScienceandTechnologyAISTMidorigaokaIkedaOsaka563-8577Japan.E-mailyasushi.shigeriaist.go.jpbDepartmentoChemistryandMaterialsEngineeringKansaiUniversityYamate-choSuitaOsaka564-8680Japan.E-mailarakkansai-u.ac.jpTheelectricalconductivityothematrixcrystalmightbeanewactortoenhancematrix-assistedlaserdesorptionionizationmassspectrometryMALDI-MSsensitivity.InMALDI-MSseveralcompoundsareusedasastandardmatrix.Utilizationosuchcompoundsisbasedonanaposterioriapproachbutthereisnotheoreticalguidanceorselectingamatrix.Inanattempttourtherunderstandperor-manceinMALDI-MSweutilizedpeptidedetectionorrandomscreeningoachemicallibrary12383compoundsorcompoundswithmatrixunctionsinMALDI-MS.Alotothiophenecompoundswereoundtobeamatrixinwhich2-5-24-dichlorobenzoyl-2-thienylaceticacidDCBTAprovidedanimportantcluetomeasuretheelectricalconductivityothematrixcrystalbecausethestructureoDCBTAisanalogoustoconductivepolymersandorganicsolarcells.Mostothecrystalsostandardmatricessuchasa-cyano-4-hydroxycinnamicacidCHCA35-dimethoxy-4-hydroxycinnamicacidsinapinicacidSAandDCBTAshowedelectricalconductiv-itywhereastheconductivityocrystalwasnotobservedin25-dihydroxybenzoicacid25-DHB.Ontheotherhandsuper-DHBusing2-hydroxy-5-methoxybenzoicacid5-methoxysalicylicacidMSAasanadditiveto25-DHBimprovedtheelectricalconductivityothecrystalthatollowedtheenhancementopeakintensityinMSspectrum.Theseobservationsmightindicatethattheelectricalconduc-tivityomatrixcrystalsisakeyconsiderationinobtainingefcientMALDIperormance.KeywordsCHCADCBTAelectricalconductivityMALDIsuper-DHBIntroductionA.Yasudaetal.Eur.J.MassSpectrom.1929372013Received16January2013nRevised7March2013nAccepted18March2013nPublication17April2013 30ElectricalConductivityofMatrixCrystalspeptideanalysesa-cyano-4-hydroxycinnamicacidCHCA535-dimethoxy-4-hydroxycinnamicacidsinapinicacidSA3and25-DHB4arerequentlyutilizedmatricesorMALDI-MSwithanitrogenlaser337nm.AlthoughmanycompoundshavebeentestedthereisnoguidelineormatrixdesigninMALDI-MS.OntheotherhandtheuseoadditivesinMALDI-MShasotenbeeninvestigatedtoimprovetheperormanceomatrixcompounds.Amongthemthe2-hydroxy-5-methoxybenzoicacid5-methoxysalicylicacidMSAaddedto25-DHBhasbeenwidelyappliedtotheanalysisopeptidesandproteins.6Theadditiono10MSAto25-DHBwasreerredtoassuper-DHB.Itwasnotclearwhysuper-DHBenhancedpeakintensityortheanalytesinMALDI-MScomparedwiththesingleappli-cationo25-DHB.ToputitbriefytheimportanceoMALDIisintheprotontransertotheanalytesinthephoto-excitedmatrixmoleculeswhenthelaserbeamisocusedontothesuraceothematrixanalytesolidsolution.HoweverithasproveddiculttounderstandMALDIionization.Theresultingionsseemtobegeneratedbymultiplemechanisms.7Asmentionedpreviouslythesearchoramatrixhasbeencarriedoutbymanyresearchers.Howeverthescaleothecompoundsseemedtobetenstohundreds.Recentlyscreeningwithapublicdiversechemicallibrarycontainingmorethan100000compoundsledtoidentiyinghitcompoundsregulatingbiologicalunctions.Thepurposeoachemicallibraryishigh-throughputscreeningordrugdiscovery.BasedonthisstrategywethoughtthatitwouldbequiteeectivetoscreenandidentiycompoundswithmatrixunctionsinMALDI-MS.MoreovertheelucidationochemicaleaturesohitcompoundsmayprovidesignicantinormationorclariyingthemechanismoMALDI-MS.Inthisstudyweexamined12383chemicalcompoundsandidentiedathiophenecompoundasagoodmatrixortheanalysisopeptidescaeineandasteroidhormoneinMALDI-MS.ThestructuraleatureoathiophenecompoundimpliedthattheelectricalconductivityothecrystalwassignicantinMALDIperormance.Herewealsoattemptedtomeasuretheelectricalconductivityomatrixcrystalorexamplewith25-DHBandsuper-DHBandacorrelationwasobservedbetweenenhancementascomparativepeptidepeakintensitiesandgenerationoelectricalconductivityocrystals.ExperimentalMaterialsAdiversechemicallibrarycontaining12383compoundsdissolvedin10mMoDMSOwasobtainedromOpenInnovationCenterorDrugDiscoverytheUniversityoTokyoTokyoJapan.The25-DHBCHCASAandMSAwereobtainedromTokyoChemicalIndustryTokyoJapan.The2-5-24-Dichlorobenzoyl-2-thienylaceticacidDCBTAmonoisotopicmass313.95711DawaspurchasedromKeyOrganicsLtdCamelordUK.PeptideNRVYVHPFhypertensionAsn1Val5-angiotensinIILSKMEDPGSVLSTACGTPGYVCaMKI165-185andphosphorylatedCaMKI165-185LSKMEDPGSVLSTpACGTPGYVweresynthesizedbyFmocpeptidechemistryusingaShimadzuPSSM-8automatedpeptidesynthesizerShimadzuKyotoJapanasdescribedpreviously.89Trypsin-digestedBSAMSstandardsanda-cyclo-dextrinwereromNewEnglandBioLabsInc.MAUSAandWakoPureChemicalIndustriesLtdOsakaJapanrespec-tively.MassspectrometryThestockstandardsolutionsopeptidescaeinetestosteroneanda-cyclodextrinwerepreparedinultrapurewater.Thestocksolutionswerethendilutedtoaconcentrationo10pmolLwith50acetonitrilecontaining0.1TFA.Aliquots1.0Lwerepipettedontoa96-wellstainless-steelMALDItargetplateBrukerDaltonicsBremenGermanyanddriedinairatroomtemperature.Forscreeningeachcompound10mM1.0LdissolvedinDMSOwasseparatelyplacedontheplateandthendriedinvacuo.MassspectrometryexperimentswereperormedinthepositiveionmodeusingaMicrofexMALDI-ToFmassspectrometerBrukerDaltonicsBremenGermanywhichwasoperatedinthelinearmodeand100lasershotssummed.InthecaseotheusualMALDI-ToFMSmeasurement10mgmL1matricesin50acetonitrilecontaining0.1TFAwereutilized.ElectrochemicalmeasurementsDisposableelectricalprintedDEPchipsmodelDEP-SP-NwereobtainedromBioDeviceTechnologyCo.IshikawaJapan.Matrixsolution2L10mgmL1in50acetonitrilecontaining0.1TFAwasdroppeddirectlyontotheDEPchipanddriedinvacuo.Thisoperationwasrepeatedatleastvetimes.TheresultingDEPchipwasconnectedtoaKeithley2400SourceMeter.Theelectricalconductivityothecrystalwasmeasuredunderaconstantvoltageat20V.ResultsanddiscussionAhitcompoundinMALDI-ToFMSTheionsignalintensityorhypertensinAsn1Val5-angiotensinIIwasprominentlyobservedromover500compoundsinadiversechemicallibrary.WhentheobservedspectrawereconinedtoasimilarsensitivitytothatoCHCAagoldstandardmatrixthenumberocandidatesasgoodmatriceswaslessthan100.Withcareulrecrystallizationocompound-samplemixtureusing50acetonitrilecontaining0.1TFAmostothesecompoundscouldexhibitgoodmolecularionsignalorhypertensinagain.Bythestructuralcomparisonothesecompoundsagreatvarietyostructureswereobserveddatanotshown.AmongthecandidatesasgoodmatricesseveralsulurcompoundswerenotableortheirstructuraleaturesFigure1andtheMALDIspectraareshowninsupportinginormationSupplementalFigures1and2. A.Yasudaetal.Eur.J.MassSpectrom.192937201331Although5-chloro-2-mercaptobenzothiazolecontainingasulfuratomhadbeenreportedasaMALDImatrixinpeptideandproteindetection10itisinfrequentlyusedforpeptideandproteinanalysisinMALDI-MSbutcurrentlyimpor-tantinglycanandoligosaccharideanalysis.116-Aza-2-thiothyminehadinitiallybeenshowntobeaneffectivematrixforanalyzingoligonucleotides12andtherewasanexamplefordetectingnoncovalentproteincomplexesbyMALDI-MS.13Inourscreeningtestwithhypertensinmonoisotopicmass1030.53489Da2-5-24-dichlorobenzoyl-2-thienylaceticacidDCBTAprovidedastrikingeffectasthematrixonMALDI-MSFigure2.Inadditiontohypertensincaffeinemonoisotopicmass194.08038Daandtestosteronemono-isotopicmass288.20892Daweretestedfortheperfor-manceofDCBTA.AsshowninFigure3DCBTAexhibitedasimilarsensitivitytothoseofCHCA.AbsorptionspectrumofDCBTAandtheresultingMALDIspectrawithDCBTAorCHCAusingtrypsindigestsofbovineserumalbuminBSAaphosphopeptidephosphorylatedCaMKI165-185anda-cyclodextrinweresummarizedinsupportinginformationSupplementalFigures3567and8.LaserdesorptionionizationmassspectrumofDCBTAwasshowninSupplementalFigure4andpolymerizationpeakofDCBTAwasnotdetectedatall.WhileitwasconcludedfromtheseobservationsthatDCBTAcouldnotreplaceCHCAasasupe-riorgoldstandardmatrixwecouldfocusourattentiononthestructureofDCBTA.DCBTAconsistsoftwomolecularfeaturesasshowninFigure1.Oneisachlorineatominthebenzenering.Theotheristhepresenceofathiophenemoiety.Recently4-chloro-a-cyanocinnamicacidCl-CCAwasreportedtobeanexcellentmatrix.14Duetoasystematicsubsti-tutionofthe4-hydroxylmoietywithachlorineatomCl-CCAwasdevelopedwithbettersensitivitythanCHCA.15FromourscreeninginadditiontoDCBTAwefoundthatseveralcompoundscontainingachlorineatomsuchasFigure1.Structureof2-5-24-dichlorobenzoyl-2-thienylaceticacidDCBTA1andotherthiophenecompounds213exhibitingmatrixfunctioninMALDI-MS. 32ElectricalConductivityofMatrixCrystals4-chloro-N-3-oxocyclohexylidenebenzohydrazideand2-24-dichlorophenyl-1-246-trihydroxyphenylethanonedemonstratedthematrixperormancewithlowerpeakintensitiesderivedromtheprotonatedmatricesdatanotshown.InMALDI-MSmeasurementwithpositiveionmodethisobservationseemstobeadvantageoussinceJaskollaetal.hypothesizedthatthelowermatrixionintensityresultsinamoreefcientprotontranserromamatrixtoanalytesinthecaseoCl-CCA.14ElectricalconductivityofcrystalAlthoughDCBTAisauniquemoleculecontainingathiophenemoietyitwasnotclearwhyDCBTAandseveralcompoundswiththiophenemoietiesexhibitedeectivematrixunctionorpeptideandproteinanalysisinMALDI-MS.Howeverwecouldinterpretthisfndinginthelightotheollowingobser-vationsiInthepastelectrodepositedpolythiopheneflmhasbeenusedorlaserdesorptioniontrapMSstudies.16iiSoltzbergandPatelhaveexploredtheuseoregioregularFigure2.MALDI-MSspectraofhypertensin10pmolusing2-5-24-dichlorobenzoyl-2-thienylaceticacidDCBTAasamatrixinscreeningfromacompoundlibrary.DCBTAwaspreparedfromDMSOaandre-crystallizedwith50acetonitrilecontaining0.1TFAb.ab A.Yasudaetal.Eur.J.MassSpectrom.192937201333Figure3.MALDI-MSspectraofahypertensinctestosteroneecaffeineusingDCBTA10mgmLandspectraofbhypertensindtestosteronefcaffeineusingCHCA10mgmL.Fivepmolofhypertensinandcaffeineand10pmoloftestosteronewereloadedonthesampleplaterespectively.Theionsmarkedwithasteriskwerederivedfrommatrixitself.abcdef 34ElectricalConductivityoMatrixCrystalspoly3-n-octylthiopheneasahighmolecularweightmatrixoranalysisosmallorganicmoleculesinMALDI-MS.1iiiThiophenehasbeenwidelyusedorthesynthesisoaconduc-tivepolymer.ivStructureoDCBTAresemblesthoseooligothiophene-containingcoumarindyesordye-sensitizedsolarcellssuchasNKX-2587andNKX-2593.1819Inthesolarcelldesignchargetransereiciencyromthedyemole-culetothenanocrystallineoxideisextremelyimportant.ThereoretheelectricalconductivityothematrixcrystalwassuspectedtobeoneotheparametersaectingthespectruminMALDI-MSthroughtheionormationprocess.HowevertheroleoelectricalconductivityothematrixcrystalhasneverbeenconsideredinMALDI-MS.Tomeasuretheelec-tricalconductivityocrystalsweutilizedDEPchipstoanalyzeeachmatrixasshowninFigure4.20Althoughtheresultingcurrentalwayschangedwithintherangeo3040pAunderconstantvoltage20Vwedetectedthesameleveloelec-tricalcurrentsromcrystalsoDCBTACHCAandSATable1.Surprisinglynocurrentwasobservedromthe25-DHBcrystal.Obviously25-DHBhasbeenusedasastandardmatrixoranalysisopeptidesproteinscarbohydratesandglycolipids.MoreoverKarasetal.reportedthattheenhancedperormanceoMALDImatrixwasobservedwiththeadditionoMSAto25-DHBasaco-matrix.6InthisstudytheelectricalconductivityincreasedbytheadditionoMSAto25-DHBasshowninTable1.Thelargestcurrentwasobservedinsuper-DHB25-DHBMSA9010largerthanthoseoMSAitselanddierentmixtureso25-DHBandMSA.NextwemeasuredMALDIspectraosuper-DHB25-DHBMSAandseveralmixtureso25-DHBandMSA.Asreportedpreviously6wealsoobservedthatsuper-DHBshowedhigherdesorptionionizationefciencythan25-DHBandMSAFigure5b.Inthecaseothemixtureo25-DHBandMSAacorrelationopeakenhancementandelectricalconductivitywasobservedFigure4.PhotographsoDEPchipsusing1DCBTA2CHCA3SA425-DHB5MSAand6super-DHB.Thegapbetweenelectrodeswascompletelyflledwitheachcrystal.MatrixCurrentpADCBTA100150CHCA100200SA10020025-DHBN.D.25-DHBMSA955100Super-DHB24-DHBMSA901015025025-DHBMSA851510015025-DHBMSA802010025-DHBMSA5050100MSA100AllmeasurementswerecarriedoutusingDEPchipsatroomtemperatureandunderaconstantvoltageat20V.FinallyacrystalonDPEchipwaspreparedundervacuum.Iacrystalabsorbedmoistureromtheairelectri-calconductionothecrystalincreasedgreatly.N.D.notdetected.Table1.Electrochemicalquantitationomatrixcrystal. A.Yasudaetal.Eur.J.MassSpectrom.192937201335intherangeoa0to20additionoMSAFigure5dandTable1.Subsequentlythereductionotheprotonatedionpeakohypertensininthepresenceo20ormoreadditionoMSAseemedtobederivedromthesuppressioneectoMSA.ThereasonsortheobservedimprovementintheMALDIspectrawithadditivesto25-DHBarelessclear.Bashiretal.reportedthatnodirectcorrelationwasobservedbetweenabsorptioncoefcientandtheabilityothemoleculetoactasthematrices25-DHBandsuper-DHB.21OntheotherhandKarasetal.suggestedthatimprovedperormanceosuper-DHBiscausedbydisturbanceothe25-DHBlattice.6Howeverthisspeculationmightnotbeaproperanswertotheincre-mentotheelectricalconductivityoacrystalbytheadditionoMSA.Duringourexperimentwesometimesobservedanincreasedelectricalconductivityothecrystalduetoabsorp-tionomoistureromtheair.ItissometimesrecognizedthatFigure5.MALDI-MSspectraohypertensinusinga25-DHBbsuper-DHBandcMSAunderthesamecondition.dDesorptionionizationefciencyohypertensinusing25-DHBmatrixinthepresenceoMSA0510152050and100.abcd 36ElectricalConductivityofMatrixCrystalsmanycompoundsgenerallyincorporatewatermoleculesintheircrystallinerameworksduringcrystallizationromthemoistsolvents.Thisimpliesthatanysignicantdierencesbetween25-DHBandsuper-DHBinMALDIperormanceandelectricalconductivityocrystalmightberelatedtocrystal-lizationwater.IntheMALDI-ToFMStheionizedsamplesareacceler-atedinanelectriceldwiththehelpoahighvoltagegridandthentheacceleratedionsareintroducedintothehighvacuumfighttube.OneotheimportantpointsinMALDI-MSmeasurementisthatthelocationotheionizingeventbeclosetothechargedplateinanionsource.ForinstanceitwasreportedthatproteinsblottedonpolyvinylidenedifuoridePVDFmembranecouldbedetectedinMALDI-ToFMSbutitssensitivitywasgreatlyreduced.22AlternativelyarelativelyhighsignalintensityoimagingmassinMALDI-ToFMSwasobservedwhencarbon-imbeddedconductivepolyethylenewasusedasaproteinblottingmembranewiththesectionedreshtissue.23SincePVDFmembraneisanelectricalinsu-latoritcouldbeconcludedthatanimprovementinsensitivityoMALDIwasproducedbytheconductivityothemembrane.TakentogetherhigherdesorptionionizationeiciencyoMALDI-MSmightbepartlyattributedtoimprovementoelec-tricalconductivityonthesuraceothetargetplateincludingthatothecrystal.OntheotherhandKimetal.recentlyreportedthatrepeti-tivelaserirradiationinMALDI-MSmadethesamplethinner.ConsequentlyincreaseinthermalconductionandlowplumetemperatureinducedinecientMALDI-MSmeasurement.2425InthecaseoourMALDI-MSmeasurementrandomwalkingmodewasusednottomakearepetitivelaserirradiationatthesamespot.HoweverurtherstudiesareneededtodeterminetherelationshipbetweenthermalconductionandelectricalconductivityinMALDI-MSmeasurement.ConclusionInthisstudyscreeningromalarge-scalecompoundlibraryinMALDI-MSwasuseultondseveralcompoundscontainingsuluratomsasmatricesorpeptides.Amongthem2-5-24-dichlorobenzoyl-2-thienylaceticacidDCBTAwasobservedasamatrixindetectionopeptidespeptidemixturesa-cyclodextrintestosteroneandcaeine.HoweverDCBTAwithathiophenemoietycouldnotexceedtheperor-manceothegoldstandardmatrixCHCA.ItisnoteworthythattheDCBTAmoleculecontainedathiophenemoietyasdodyesororganicsolarcells.ThisobservationledtothemeasurementotheelectricalconductivityothecrystalusedorMALDI-MS.MostcrystalsostandardmatricesDCBTAandsuper-DHBpossessedelectricalconductivitywhereasanelectricalconductivityo25-DHBcouldnotbedetected.Oneadvantagesosuper-DHBseemstobeanelectricalconduc-tivityothecrystal.FurtherstudiesareneededtodeterminetherelationshipbetweenthesensitivityoMALDI-MSandtheelectricalconductivityothematrix.HoweverourresultsmaysuggestthattheelectricalconductivityomatrixcompoundsisimportantandurtherexaminationothiseaturewillcontributetoabetterunderstandingotheMALDIprocessandmethod.AcknowledgmentsWethankDrLeslieSargentJonesAppalachianStateUniversityorcareulreadingothemanuscript.WearegrateultoDrMakotoNakataPeptideInstituteInc.DrHidenoriNagaiandDrNobutakaTanigakiAISTorvaluablediscussions.ThisworkwassupportedbytheAISTresearchgrantandbyaGrant-in-AidorscienticresearchromJapanSocietyorthePromotionoScienceNo.22350040toR.A.ThisworkwasalsosupportedbyPlatormorDrugDiscoveryInormaticsandStructuralLieScienceromtheMinistryoEducationCultureSportsScienceandTechnologyJapan.SupportinginformationSupportinginormationmaybeoundintheonlineversionothisarticle.References1.K.TanakaH.WakiY.IdoS.AkitaY.YoshidaandT.YoshidaProteinandpolymeranalysesuptomz100000bylaserionizationtime-ofightmassspectrom-etryRapidCommun.MassSpectrom.21511988.doi10.1002rcm.12900208022.M.KarasandF.HillenkampLaserdesorptionioniza-tionoproteinswithmolecularmassexceeding10000DaltonsAnal.Chem.6022991988.doi10.1021ac00171a0283.R.C.BeavisandB.T.ChaitCinnamicacidderivativesasmatricesorultravioletlaserdesorptionmassspec-trometryoproteinsRapidCommun.MassSpectrom.34321989.doi10.1002rcm.12900312074.K.StrupatM.KarasandF.Hillenkamp25-Dihydroxybenzoicacidanewmatrixorlaserdesorption-ionizationmassspectrometryInt.J.MassSpectrom.111891991.doi10.10160168-11769185050-V5.R.C.BeavisT.ChaudharyandB.T.Chaita-Cyano-4-hydroxycinnamicacidasamatrixormatrix-assistedlaserdesorptionmassspectrometryOrg.MassSpectrom.271561992.doi10.1002oms.12102702176.M.KarasH.EhringE.NordhoB.StahlK.StrupatF.HillenkampM.GrehlandB.KrebsMatrix-assistedlaserdesorptionionizationmassspectrom-etrywithadditivesto25-DihydroxybenzoicacidOrg.MassSpectrom.2814761993.doi10.1002oms.1210281219 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39ISSN1469-0667IMPublicationsLLP2013doi10.1255ejms.1209AllrightsreservedEUROPEANJOURNALOFMASSSPECTROMETRYMALDIhasbeenestablishedasaversatileandpracticaltoolfortheanalysisofmanydifferentclassesofcompoundsproteins12polymers34lipids56peptides17andoligonucleotides89butalsosmallmoleculessuchasmetabolitesanddrugs.1012Itfeatureshighsensitivityhightoleranceforsaltsandcontami-nantsandagenerallystraightforwardsamplepreparation.Howeveranalysisinthelowmzregionremainschallengingduetothepresenceofmatrix-relatedionsinterferingwithpeaksofanalyte-relatedions.Therehavebeenconsider-ableeffortstoworkaroundthislimitation.Forinstancethematrix18-bisdimethylaminonaphthaleneprotonspongewasdemonstratedtoexhibitaverylowchemicalbackgroundalthoughitrequiresa11analytematrixratio.13Matrixsuppressioneffects1415havebeenreportedtoleadtoconsid-erablycleanerspectra.Alsomatriceswithmolecularweightshigherthan1000mzhavebeenreported.1617Matrix-freesubstratessuchasdesorptionionizationonsiliconDIOSprovidecleanbackgroundsbutchipsthatgiveveryhighsensi-tivityaretimeconsumingtooptimizeandhavethereforenotgainedwidespreadapplication.1819FornegativeMALDIofsmallmolecules9AAhasbeendemonstratedbyseveralgroupstoproduceonlyfewbackgroundpeakswhileprovidingdetectionofmanyphosphorylatedmetaboliteswithveryhighsensitivitydowntoattomoles.2021Howeversinceitisnotentirelyfreeofbackgroundpeaks9AAmaynotbetherightchoicefortheanalysisofcertaincompoundssuchasglucuronicacidforwhichthemassdifferenceofthedeprotonatedmoleculariontothemolecularionofthematrixisonly0.05Da.Also9AAisnotequallyeffectiveforallacidicmetabolites.Forinstanceaminoacidsarenotefficientlyionizedby9AA.Thereforealternativematriceswithcomparablesensitivitybutadifferentdetectionrangeandwithdifferentback-groundpeaksaredesirable.AsimilarstudybyPortaetal.hasalreadybeenpresentedforpossiblealternativestoMatrix-assistedlaserdesorptionionizationmatricesfornegativemodemetabolomicsStephanR.FagereraSimoneNielsenaAlfredoIbezandRenatoZenobiDepartmentofChemistryandAppliedBiosciencesETHZurichCH-8093ZurichSwitzerland.E-mailzenobiorg.chem.ethz.chMatrix-assistedlaserdesorptionionizationMALDIhasbeenshowntobehighlysensitiveforanalyzinglow-masscompoundssuchasmetabolitesiftherightmatrixisused.9-aminoacridine9AAisthemostcommonlyemployedmatrixfornegativemodeMALDI-MSinmetabolomics.Howevermatrixinterferencesandthestronglyvaryingsensitivityfordifferentmetabolitesmakeasearchforalternativematricesdesirableinordertoidentifycompoundswithadifferentchemicalbackgroundandorfavoringadifferentrangeofanalytes.WetestedtheperformanceofaseriesofpotentialnegativemodeMALDImatriceswithamixof29metabolitescontainingaminoacidsnucleotidephosphatesandKrebscycleintermediates.WhileethacridinelactatewasfoundtoprovidelimitsofdetectionLODsinthelowfemtomolerangefornucleotidephosphatesaminoacidsandKrebscycleintermediatesinthelowpicomolerange4-amino-2-methylquinolineshowedLODsinthepicomolerangeformostmetabolitesbutiscapableofionizingabroaderrangeofanalytesthanboth9AAandethacridine.KeywordsmetabolomicsnegativemodeMALDIMALDImatrixscreening9-aminoacridineIntroductionS.R.Fagereretal.Eur.J.MassSpectrom.1939472013Received27October2012nRevised6February2013nAccepted19February2013nPublication4March2013acontributedequally 40MALDIMatricesforNegative-ModeMetabolomicsthepositive-modematrixa-cyano-4-hydroxycinnamicacidCHCA.22Herewescreened20dierentnitrogencontaininghetero-cycliccompoundswithtwo-andthree-memberedaromaticringsortheirsuitabilitytoreplace9AAintheseinstances.WewilldiscusstheirphysicochemicalpropertiesandtheLODsreachedorthetestedmetabolitemix.ExperimentalChemicalsMetabolitesTheaminoacidsolutionAAS18containingL-alanineL-arginineL-aspartateL-cystineL-glutamicacidglycineL-histidineL-isoleucineL-leucineL-lysineL-methionineL-phenylalanineL-prolineL-serineL-threonineL-tyrosineandL-valineataconcentrationo1.25mMoreachaminoacidwaspurchasedromSigmaAldrichBuchsSwitzerland.Adenosinemonophosphateructose-16-bisphosphatecytidine-5-monophosphatedisodiumsaltadenosinetriphosphatedisodiumsaltguanosine-5-diphosphatedisodiumsaltandmethanolwerealsoromSigmaAldrichBuchsSwitzerland.Fumaricacidmalicacidpyruvicaciduridine-5-monophosphatedisodiumsaltandphosphoenolpyruvicacidmonopotassiumsaltwereromABCRKarlsruheGermany.Glucose-1-phosphatedisodiumsalttetrahydratewasromFlukaBuchsSwitzerland.CoenzymeAwasromApplichemDarmstadtGermany.AcetonephotospectrometricalgradewasboughtromAcrosWohlenSwitzerland.Matrices6-aminoquinoline9-amino-1234-tetrahydroacridine9-aminoacridinehydrochloridehemihydrate36-diamino-acridine3-aminoquinolineethacridinelactate18-bisdimethylaminonaphthaleneprotonspongeberberinechloridecrystalvioletandbiquinolinewerepurchasedromSigmaAldrichBuchsSwitzerland.QuinacrineandphenanthrolinewereromAcrosWohlenSwitzerland.1-aminonapthalenewasboughtromFluka.4-aminoquinolinewasboughtromBioblocksSanDiegoCaliorniaUSA.25-DihydroxybenzoicacidwaspurchasedromFlukaBuchsSwitzerlandandpurifedbyrecrystallization.Ethacridinereebasewasliberatedbymixing1goethacridinelactatein100mLoH2Owith3gosodiumhydroxidein10mLoH2O.Theyellowprecipitatewasflteredandwashed10timeswithapproximately30mLoH2O.PreparationothemetabolitemixAmetabolitemixwithaconcentrationo1mMoreachcompoundwasprepared.Itcontained18aminoacidsfvenucleotidephosphatesaswellasphosphoenolpyruvateructose-16-bisphosphatecoenzymeApyruvateumarateandmalateseeTable1orcompletelistandmassothedeprotonatedormMHTestedmatricesTwentytwo-andthree-memberedheterocycliccompoundscontainingnitrogenatomswerechosenaspotentialMALDImatricesTable2.Allwerepurchasedromcommer-cialsources.Allmatricesweredissolvedinmethanolat10mgmL1exceptor9-aminoacridine95methanol5H2Oethacridinelactate80methanol20H2Oandethacridineacetoneduetolowsolubilityinpuremethanol.SamplepreparationAserialdilutionothemetabolitemixivedilutionsrom50Mto5nMwithaactoro10betweenoneandthenextwaspreparedandmixed11vvwiththerespectivematrix.Then0.5Lothatmixwerespottedontoastainlesssteel384-wellMALDItarget12381mmABSciexatatempera-tureo50Csixreplicateseach.ThehighertemperaturewasMetaboliteMHmonoisotopicmassGlycine74.0248Pyruvate87.0088Alanine88.0404Threonine102.0561Serine104.0353Proline114.0561Fumarate115.0037Valine116.0717Leucine130.0874Aspartate132.0302Malate133.0142Lysine145.0983Glutamate146.0459Methionine148.0438Histidine154.0622Phenylalanine164.0717Phosphoenolpyruvate166.9751Arginine173.1044Tyrosine180.0666Cystine239.0166Glucose-1-phosphate259.0224Uridinemonophosphate323.0286Fructose-16-bisphosphate338.9888Adenosinemonophosphate346.0558Guanosinediphosphate442.0171Cytidinetriphosphate481.9772Adenosinetriphosphate505.9885CoenzymeA766.1079Table1.Listofusedmetabolitesandrespectivemassofdeprotonatedmolecules. S.R.Fagereretal.Eur.J.MassSpectrom.193947201341chosentoincreasethespeedocrystallizationwhichislinkedtothesizeandhomogeneityothecrystalbedseeFigure1.At50Cnoincreaseodegradationproductsrommetaboliteswasobserved.UVvismeasurementsUVvisspectrawererecordedonaThermoGenesys10Sinsolutionrom300nmto900nm.Extinctioncoecientsatawavelengtho355nmweredeterminedusingconcentrationsbetween250Mand500Moeachmatrixin50methanolwater.Thentheconcentrationothemotherliquorinsatu-ratedsolutionswasdetermined.MALDImassspectrometryMeasurementswereperormedonatandemtimeofightToFToFinstrument4800plusABSciex.TheNdYAGlaser355nmhadacircularspotsizeo30mindiameteratarepe-titionrateo200Hz.Laserintensityoptimaoramaximumosignal-to-noiseweredeterminedindividuallyoreachmatrix.Foreachinalspectrum600shotswereaccumulated20shotseachat30dierentrandomizedpositions.Anedge-biasedlaserrastercouldpotentiallyimprovethesignalinten-sityormatriceswitharing-shapedcrystallizationpatternbutwechosetouseastandardizedhomogeneoussearchpatternorallcompounds.DataprocessingDatawererecordedwiththeinstrumentsotware4000SeriesExplorerandthenexportedtotheABSciexDataExplorersotwareasT2Diles.ThentheseileswereonceagainexportedtoASCIIlesandurthertreatedinMATLABr2011aMathworkstoextractthepeakheightsandorstatisticalanalysis.Limitsodetectionweredeterminedorasignal-to-noiseratio3.DistinguishingchemicalbackgroundromanalytesignalswasachievedbyitestingwhetherthesignalriseswithincreasingconcentrationothemetabolitemixusingMATLABplotsnotshownandiiaccuratemassmeasurementsspectrawerecalibratedrstexternallyandtheninternally.ResultsanddiscussionChoiceofcompoundsWebasedourchoiceonthesizeothearomaticsystemtwoorthreeringsandtherequirementornitrogenatomsasprotonacceptinggroups.Therstcriterionisrelatedtotheeectiveabsorptionothelaserwavelength.Thenitrogenatomseitherintheringorasanaminounctionalityshouldensuretheabstractionoaprotonromtheanalyteduringtheionizationprocessi.e.thematrixmoleculeshouldeatureahighpKavalueotheconjugatedacid.ThisrequirementcanbederivedromthecurrentmodelsorMALDIionization.Theluckysurvivormodelpostulatesthattheanalytemoleculesarealreadychargedinsolutionandconservedinthisormwhenincorporatedintothematrixcrystals.2526ThismodelsuggeststhatmatriceswithhighpKavaluesarebeneicialasmoreextensivedeprotonationotheanalyteswouldincreasethenumberosurvivorsollowingneutralizationprocessintheplume.Thephotoionizationmodelormulatedorpositivemode27statesthationizationoccurspredominantlyintheablationplumewhereionsarecreatedviaprotontransertomatrixionsormedviaenergypooling2528.Inourcaseusingnegativemodeahighprotonanityothematrixanioniscrucialorahighyieldochargedanalytes.Forpeptidesithasbeenshownthatbothmodelsmayapplysimultaneouslydependingonvariousparameterssuchassizeothepeptide.29Inconclusionorbothmodelstheabilityoamatrix-derivedspeciestoabstractaprotonromtheanalyteeitherinsolutionorinthegasphaseisimportantortheeciencyothematrix.AminoacridinesandaminoquinolineswithpKavalueshigherthanthato9AAwerechoseninordertotestwhethersolutionpKahasadirectinfuenceonionizationeciency.Ithisisnotthecasethiswouldindicatethatdeprotonationismorelikelytooccurinthegasphasewheretheabilityoamatrix-derivedspeciestoabstractaprotonmaybedierentromthatinsolution.Converselywewerealsointerestedwhethereec-tiveionizationcanbeachievedwithlessbasicmatricesorexampleacridine.EvaluationofthesensitivityandanalytecoverageThelimitsodetectionoraselectionothematricesthatperormedbestorouranalytemixaregiveninTable3.TherestisreportedinthesupplementalinormationTableS1.ThebackgroundspectraoallusedmatricesareshowninFigureS1.Byarthelowestlimitsodetectionwerereachedortheanalysisonucleotidephosphateswith9AAwhichrendersthismatrixsuitableorapplicationswhereonlyaminuteamountosuchmaterialispresent.3031AdenosinetriphosphateandcoenzymeAorexamplewerestilldetectedatthe125attomolelevel.Yetonlyiveaminoacidswereobservablewith9AAasthematrix.AlsononeotheKrebscycleintermediatesweredetected.IncontrastinspectrarecordedwithethacridinelactateATPandcoenzymeAwereonlydetectedatalevelabove12.5emtomolesbut12outoFigure1.Crystallizationqualityof9-aminoacridineatroomtemperatureleftand50Cright. 42MALDIMatricesforNegative-ModeMetabolomicsNameStructureMWgmol1e355Lmol1cm1pKaBackgroundqualityacridine179.22113105.60239-aminoacridine194.266539.514-amino-2-methylquinoline158.203729.40.5-1-aminonaphthalene143.195724.20.1-3-aminoquinoline144.1735204.344-aminoquinoline144.172219.1724o6-aminoquinoline144.1734235.06o9-amino-1234-tetrahydroacridine198.262969.60.2Berberine371.8124736na22-biquinoline256.30102.20.6-cinchonidine294.3909.30.7oTable2.Listofmatriceswiththeirchemicalstructures.Thenumberofchemicalbackgroundpeaksisindicatedbythecategoriesveryfewbackgroundsignalscomparableto9AAoacceptablecomparabletoDHBinpositivemodeatalaserirradianceof6.4108Wcm2interferenceisworsethanDHBinpositivemodeatalaserirradianceof6.4108Wcm2.ThesolutionphasepKavaluesweretakenfromtheliteratureReferences23and24determinedbytitrationin50methanolwaterindicatedbytheasteriskorelsecalculatedusingAdvancedChemistryDevelopmentACDLabsSoftwareV11.021994-2013ACDLabs.ThesolutionpKaofethacridinelactatereferstothesameequilibriumasthatofethacridinei.e.betweenthesinglyandnon-protonatedform. S.R.Fagereretal.Eur.J.MassSpectrom.19394720134317aminoacidswereound.Itwassurprisingthatthelactateormwhichdriesmoreslowlyduetothehigherwatercontentthatwasnecessaryinordertodissolveitandthering-shapedcrystallizationpatternthatresultedperormedconsiderablybetterthanthereebasewhichgaveamorehomogeneouscrystalbed.36-diaminoacridineenableddetectiono15outo17aminoacidsbutonlyoewnucleotidephosphates.Whileacridineperormsworsethan9AAintermsosensitivityLODvaluesinthepmolrangeormostmetabolitesitisauniversallyapplicablematrixorthetestedanalytes.Itsapplicabilityinnegativeionmodeissurprisingsinceitisaveryweakbaseinsolution.Thepositivelychargedmatricesberberineandcrystalvioletalsoalloworthedetectiononegativeions.Thesefndingsindicatethattheionizationoanalytesislikelytooccurinthegasphasei.e.thatprotonabstractionbyamatrix-derivedgas-phasespeciesismoreeective.Amongallthetestedquinolines4-amino-2-methyl-quinolineshowedthebestanalytecoverage.Elevenaminoacidsandouroutofvenucleotidephosphatesweredetected.Thequestionarisesastowhy9AAandethacridinelactateperormmuchbetterorphosphorylatedanalytesthantherestothetestedmatrices.IngeneralthereishardlyanyliteratureaboutthestructurereactivityrelationshipoMALDImatricesespeciallyorthenegativeionmode.BothNameStructureMWgmol1e355Lmol1cm1pKaBackgroundqualityCrystalviolet407.982143na36-diaminoacridine209.2521029.52325-dihydrobenzoicacid154.1223622.972429-dimethyl-110-phenanthroline208.26336.00.3oethacridine253.30522811.0423ethacridinelactate343.151650011.0423o110-phenanthroline180.2195.20.1oprotonsponge214.3112.1quinacrine413.98303910.50.3 44MALDIMatricesforNegative-ModeMetabolomicsJaskollaetal.aswellasShroetal.claimthattheprotonafnityisdecisiveorthequalityoamatrix.3233Morespecif-callyaccordingtoJaskollanotonlytheionizationotheanalyteiscriticalbutalsotheabsenceoneutralizationreactionswiththematrixi.e.thereversalotheioniza-tionreactionwhichbothgreatlybeneitromaverylowprotonafnityothematrixinpositivemodeandaveryhighprotonafnityothematrixinnegativemode.WhiletuningtheprotonafnitywasidentifedasaguidingprincipletoimproveexistingmatricesdemonstratedorCHCAbythesynthesiso4-chloro-a-cyanocinnamicacidthispropertyalonecannotexplainthedierencesinperormance.Thematrix18-bisdimethylaminonaphthaleneprotonspongeorinstancehasahigherprotonainity1028kJmol1at298K34than9AA977kJmol1at298K35butperormedpoorlywiththetestedanalytemix.Insteadacombinationoseveralphysicochemicalpropertiesseemstomaketheaminoacridines9AAandethacridinelactatesuitableorthedetectionoverylowquantitiesophosphorylatednucleotides.TheaminogroupatPosition9isessential.Thiscanbederivedromtheactthattheotherwiseidenticalacridineisineriortoanyotheaminoacridines.Itisworthnotingthattheperormanceothelactatesaltoethacridinesurpassedthatothereeorm.Possiblyanalyteincorporationwasmoreefcientinthecaseotheioniccompoundduetotheincreasedormationoionpairsoorexamplephosphorylatedmetaboliteswiththeprotonatedethacridine.Analyte9-amino-acridineEthacridinelactate36-diaminoacridine4-amino-2-methylquinolineAcridineQuinacrineCrystalvioletGlycine12501250Pyruvate12501250012500Alanine1250125001250Threonine1250125001250Serine1250012501250125012501250Proline125012501250012500Fumarate12501250125012501250125Valine1250012501250125001250Leucine12500125012501250125012500Aspartate125001251251250125012501250Malate125012501250125012501250Lysine125001250012501250012500Glutamate12501251250012501250125001250Methionine125012501250012501250125012500Histidine12500125012501250125012501250Phenylalanine12501250012501250012500Phosphoenolpyruvate12.51.251250125012500125012500Arginine1250125001250012500Tyrosine1250012512501250Cystine12512500125001250012500Glucose-1-phosphate0.1251.2512501250125001251250Uridinemonophosphate0.1251.251250125012501251250Fructose-16-bisphosphate1.2512512501250Adenosinemonophosphate1.251251250125125012501250Guanosinediphosphate1.251.251250125001250Cytidinetriphosphate1.251.251250Adenosinetriphosphate0.12512.51250125001250CoenzymeA0.12512.512501250Table3.Limitsofdetectionforeachmatrixandmetabolitecombinationinfmol.Theasteriskindicatesmatrixinterference.Ablankspacemeansthatthemetabolitewasnotdetected. S.R.Fagereretal.Eur.J.MassSpectrom.193947201345Wetested9-amino-1234-tetrahydroacridinewhichisidenticalto9AAexceptorthehydrogenatedormooneothesiderings.Wealsotested4-aminoquinolinewhichisalsocloselyrelatedto9AAbutismissingonearomaticsidering.Neitheryieldasintensenucleotidephosphateanionsignalsaseicientlyastheaminoacridinesprobablyduetoaloweranalyteionizationandorincorporationefciency.Thisindicatesthat-stackingotheacridineringwiththenucleotidebasescouldoccurandimproveco-crystallization.Homogeneityocrystallizationisalsoanimportantactorbutimpossibletopredictromthechemicalstructure.9AAgivestheinestcrystalsandmosthomogeneouscrystalsSupplementalFigureS3.OtherpossibleactorssuchassolubilityothematrixinthesolventusedorthehydrophobicityothematrixindicatedbythedistributioncoefcientlogDweredismissedsincenoclearcorrelationwiththeperormancewasobserveddatanotshown.Anotherinterestingquestioniswhy9AAperormsbetterthanethacridinelactateintheanalysisophosphorylatedcompoundsbutworseoraminoacids.ForoneethacridineismorebasicsolutionpKao11.2vs9.7whichisapparentlynotcrucialorthealreadypreormedphosphorylatedionsbutmoresoorzwitter-ionicaminoacids.ThelattertypicallyeaturepKavalueso9to10orthea-aminogroupwhichisinthesamerangeasthepKao9AA.Aactorthatdecreasestheeectivenessoethacridineisahighdegreeoin-sourceragmentation.Thepeakatmz224.089isamongthestrongestsignalsinthespectrumcorrespondingtothelossothealkylchainotheethoxyresidue.Ingeneralthespectrumoethacridinelactatecontainsmorechemicalbackgroundpeaksthanthato9AA.AccordingtooneothemodelsoMALDIionormation25collisionsomatrixmoleculesintheexcitedstateleadtoenergypooling.Someractionomatrixmoleculessurpassesthepotentialthatisnecessaryoranalyteionization.Howeveriacompetingragmentationreactionconsumesaconsiderableamountoenergytheefciencyothepoolingprocessandthere-oretheionizationefciencyacrosstheboardisloweredwhichwebelievetobethecasehere.Furthermoreitispossiblethattheproductotheethylenelossoethacridineanalcoholactsasaprotondonorinthegasphase.Theethacridinealcoholcanneutralizeanalyteanionsbyprotontranserromitshydroxylgroupsincetheresultingalcoho-lateisstabilizedbymesomerism.Asaresulttheyieldoanalyteanionsdecreases.PhysicochemicalcharacterizationoftheinvestigatedmatricesMatricesornegativemodemustpossessseveralqualitiesinordertoworkefciently1theymustabsorbtheappliedlaserwavelengthtypically337nmor355nm2thecrystalbedshouldbehomogeneouslydistributedacrosstheMALDIwellinordertopreventsweetspotphenomenaandgeneratereproduciblespectra3dependingonthetheoryoionproductionthematrixshouldeithereatureahighsolutionphasebasicityluckysurvivortheory36orgasphasebasicitygas-phasechargetransermodel.27Thesethreepointswillbeaddressedintheollowingparagraphs.UVabsorptionat337nmand355nmWedeterminedtheextinctioncoefcientsinsolutionTable2theullspectraareshowninSupplementalFigureS2asanapproximatemeasureortheabsorptioninthesolidstateorwhichabsorptionbandsareexpectedtobebroader.Amongthemoststronglyabsorbingmatricesareacridineethacridineandberberinewithextinctioncoeicientssurpassing10000Lmolcm1.Themajorityomatricesorexamplealltheaminoquinolineshaveevaluesaround20004000Lmolcm1.Unexpectedlydespitetheirconjugatedringsystemstwoorthreememberedphenanthrolineandcinchonidineabsorbpoorlyat355nm.Apossibleexplanationorphenanthrolineisthatsterichindrancelonepaircollisionmighttwistitssp2-systemoutoplaneandthereoreblueshittheabsorptionbands.Cinchonidineabsorbspoorlyinitsneutralorm.379-aminoacridinehasanextinctioncoefcientoonly653Lmolcm1whichgiventhatitisaveryeectivematrixsuggeststhatevenalowerevalueissufcientandthereorenotalimitingactor.CrystallizationMicroscopeimagesomatrixcrystalsareshowninthesupplementalinormationFigureS3.Fourcategoriesocrystallizationpatternswereobservedorthetestedmatricesauniormcrystalbedscouldbeachievedor9-aminoacridine9-amino-1234-tetrahydroacridineandethacridinereebasebringsocrystalsresultedromspottingquinacrineethacridinelactate36-diaminoacr-idine4-amino-2-methylquinolineandprotonspongecnon-uniormnon-symmetricalcrystalspatcheswereobservedoracridine6-aminoquinoline22-biquinolineand-cinchonidine.Finallydflmswereormedby34and5-aminoquinoline110-phenanthroline29-dimethyl-110-phenanthroline.Onewouldassumethehomogeneouscrystalbedoorexampleethacridinereebasewouldgivebetterresultsthanthecrystalringthatwasobservedorexampleorethacridinelactate.Howevertheexperimentaldataindi-catedtheoppositeinthiscaseseeLODsinTable3.Herethesignalsthatwererecordedinthesweetspotsotheringwereclearlysuperiortothesignalosmalluniormcrystals.Asalreadysuggestedabovewebelievethatthehighersensitivityothesaltormisduetobetteranalyteincorporation.InFigure2relativestandarddeviationsotheachievedintensitiesorumarateandaspartateusing9AAacri-dineethacridinelactatequinacrine36-diaminoacridine4-amino-2-methylquinolineandcrystalvioletareshown.Thesemetaboliteswerechosenbecausetheywereionizedbyallotheshownmatricesexceptumarateor9AA.Thestandarddeviationsareallbetween7and42exceptor104or9AAwhichiscommonorMALDIapplications.Thestandarddeviationothesignalintensityusingmatriceswith 46MALDIMatricesforNegative-ModeMetabolomicsring-shapedcrystallizationpatternswasnotineriortothemorehomogeneouspatternso9AAandacridinewhichissurprising.ConclusionsWetestedtwentypotentialmatricesornegativemodeMALDI.While9-aminoacridinewasconrmedasthematrixwiththebestperormanceorphosphorylatedcompoundsweoundthatespeciallyethacridinelactateand4-amino-2-methylquinolineshouldbeconsideredwhenaminoacidsorganicacidsorexampleromthecitratecycleaswellasnucleotidephosphateshouldbedetectedinthesamemixtureandquantitiesarenotbelowthepmolerange.Theactthatmetabolitescanbedetectedwithmatriceswithonlymodestbasicityindicatesthatthisclassocompoundsmayionizepredominantlyinthegasphaseinsteadoexistingaspreormedionsinthecrystalbed.AcknowledgmentThisprojectwasco-nancedbytheSwissKTIKommissionrTechnologieundInnovationgrantno.13123.1PFNM-NM.References1F.HillenkampandM.KarasMassspectrometryopeptidesandproteinsbymatrix-assistedultravioletlaserdesorptionionizationMethodsEnzymol.1932801990.doi10.10160076-68799093420-P2M.KarasandF.HillenkampLaserdesorptionioniza-tionoproteinswithmolecularmassesexceeding10000daltonsAnal.Chem.602299-23011988.doi10.1021ac00171a0283D.C.SchriemerandL.LiDetectionohighmolecularweightnarrowpolydispersepolymersupto1.5milliondaltonsbyMALDImassspectrometryAnal.Chem.6827211996.doi10.1021ac960442m4U.BahrA.DeppeM.KarasF.HillenkampandU.GiessmannMass-spectrometryosynthetic-polymersbyUVmatrix-assistedlaserdesorptionionizationAnal.Chem.6428661992.doi10.1021ac00046a0365D.J.HarveyMatrix-assistedlaser-desorptionionizationmass-spectrometryophospholipidsJ.MassSpectrom.3013331995.doi10.1002jms.11903009186J.SchillerJ.ArnholdS.BenardM.MllerS.ReichlandK.ArnoldLipidanalysisbymatrix-assistedlaserdesorptionandionizationmassspectrometryAmethodologicalapproachAnal.Biochem.267461999.doi10.1006abio.1998.30017A.ShevchenkoM.WilmO.VormandM.MannMassspectrometricsequencingoproteinssilver-stainedpolyacrylamidegelsAnal.Chem.688501996.doi10.1021ac950914h8E.NordhoA.IngendohR.CramerA.OverbergB.StahlM.KarasF.HillenkampP.F.CrainandB.ChaitMatrix-assistedlaserdesorptionionizationmassspectrometryonucleicacidswithwavelengthsintheultravioletandinraredRapidCommun.MassSpectrom.67711992.doi10.1002rcm.12900612129U.PielesW.ZurcherM.ScharandH.E.MoserMatrix-assistedlaser-desorptionionizationtime-o-fightmass-spectrometryapowerultoolorthemassandsequence-analysisonaturalandmodiedoligo-nucleotides.NucleicAcidsRes.2131911993.doi10.1093nar21.14.319110L.H.CohenandA.I.GusevSmallmoleculeanalysisbyMALDImassspectrometryAnal.Bioanal.Chem.3735712002.doi10.1007s00216-002-1321-z11A.NordstromE.WantT.NorthenJ.LehtioandG.SiuzdakMultipleionizationmassspectrometrystrat-egyusedtorevealthecomplexityometabolomicsAnal.Chem.804212008.doi10.1021Ac701982e12S.Khatib-ShahidiM.AnderssonJ.L.HermanT.A.GillespieandR.M.CaprioliDirectmolecularanalysisowhole-bodyanimaltissuesectionsbyimagingMALDImassspectrometryAnal.Chem.7864482006.doi10.1021Ac060788p13R.ShroL.RulisekJ.DoubskyandA.SvatosAcid-base-drivenmatrix-assistedmassspectrometryortar-getedmetabolomicsProc.Nat.Acad.Sci.USA106100922009.doi10.1073Pnas.090091410614Z.GuoandL.HeAbinarymatrixorbackgroundsuppressioninMALDI-MSosmallmoleculesAnal.Bioanal.Chem.38719392007.doi10.1007s00216-006-1100-315G.McCombieandR.KnochenmussSmall-moleculeMALDIusingthematrixsuppressioneecttoreduceor020406080100120RelativeStandardDeviationinPercentFumarateAspartate9-aminoacridineacridineethacridinelactatequinacrine36-diaminoacridineCrystalviolet4-amino-2-methylquinolineFigure2.Signalreproducibilityoffumarateandaspartateusingdifferentmatrices.Fumaratewasnotdetectedwith9AA. 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49ISSN1469-0667IMPublicationsLLP2013doi10.1255ejms.1211AllrightsreservedEUROPEANJOURNALOFMASSSPECTROMETRYFlavonoidsareawidespreadgrouponaturalproductsormedassecondaryplantmetabolitesandhavediversebiologicalunctionsandbenecialproperties.1Thedominantfavonoidsnaturallyoccurringinplantsandmedicinalherbsarefavonoidglycosidesandtheuseothesecompoundsinoodstusandnutraceuticalshasrecentlyreceivedmuchattention.Flavonoidglycosidesareconsideredtobemajorbioactiveconstituentsomedicinalplantswhichhavepotentialbenetsorhumanhealthincludingantioxidantantitumorandradicalscavengingactivities.26Theyarealsoreportedtohaveotherbiologicalactivitiesincludingcardiovascularprotectionandanti-infammatoryandanti-viraleects.79Ultra-high-perormanceliquidchromatographyelectrosprayionizationtandemmassspectrometryUHPLCESI-MSMShasbeenusedeectivelyortheidenticationofavonoidglycosidesrommedicinalplants.1012Thistechniqueenablesragmentationanalysiswithaccuratemassmeasure-mentandtheelucidationocharacteristicragmentationpathwaysotargetandornon-targetcompounds.Therag-mentationpatternsofavonoidshavebeenextensivelyinves-tigatedandmanyrulesorstructuralidenticationhavebeenFragmentationcharacterizationanddierentiationoisomericdiglycosyllavonoidsusingultra-high-perormanceliquidchromatographyelectrosprayionizationquadrupoletime-o-lighttandemmassspectrometryinnegativeionmodeXueengGuoYongdeYueFengTangJiaSunJinWangXiYaoandHangXunStateForestryAdministrationKeyOpenLaboratoryInternationalCentreorBambooandRattanBeijing100102PRChina.E-mailyueydicbr.ac.cngx71622icbr.ac.cnFlavonoidsexhibitawiderangeobiologicalactivitiesandtheragmentationcharacterizationanddierentiationoisomericdiglycosylfavonoidsisanimportantareaoresearch.Inthisstudyultra-high-perormanceliquidchromatographyelectrosprayionizationquad-rupoletime-o-fighttandemmassspectrometrywasusedtoidentiytwopairsoisomericdiglycosylfavonoidsnaringinnarirutinandneohesperidinhesperidin.ThetwopairsofavonoidswereidentiedbyproductiontandemmassspectraotheprecursorionsMH.Theresultsshowedthatcharacteristicproductionsdistinguishingfavonoidsglycosylatedwithrhamnosyl-a12-glucosylromisomericfavonoidsglycosylatedwithrhamnosyl-a16-glucosylweretheprecursorionMHanditscharacteristicproductionsY0and02X0H.KeywordsUHPLCESI-Q-ToF-MSMSisomericdiglycosylfavonoidsragmentationcharacterizationIntroductionX.Guoetal.Eur.J.MassSpectrom.1949552013Received24January2013nRevised22February2013nAccepted25February2013nPublication26February2013 50FragmentationCharacterizationandDierentiationoIsomericDiglycosylFlavonoidsestablishedandusedorthecharacterizationounknowncompounds.1315UHPLCESI-MSMShasproventobeauseultechniquehavinghighsensitivityandresolution.ThemajorlimitationoMSorMSMSinstructuralelucida-tionisthedicultyindierentiatingamongisomers.Therehavebeenmanyreportsotheragmentationcharacteristicsofavonoidglycosideisomersusingtandemmassspectro-metry.1618Severalrulesbasedonvariousionintensityratioshavebeenproposedandappliedtothecharacterizationofavonoidglycosideisomers.ForexamplethediagnosticionsY0andY0Hhavebeensuggestedtocharacterizetheposi-tionoglycosylationandtheaglyconenature.1920Flavonoidsglycosylatedwithrhamnosyl-a12-glucosylorrhamnosyl-a16-glucosylarethemostcommonfavonoiddiglycosides.Someotheseoccurinhighconcentrationsinruitsothecitrusgenusandareimportantaschemo-markers.21Theseisomersonlydierintheinter-glycosidiclinkagetypebetweenthetwomonosaccharides.DierencesbetweentheseconigurationshavebeenreportedtobeuseulorcharacterizingO-diglycosylfavonoidisomers2223buttherehavebeenewsystematicinvestigationsothecharacteristicproductionsoisomericdiglycosylfavonoidsusingquadrupoletime-o-fightQ-ToFMSMS.ThepurposeothepresentstudywastoinvestigatetheragmentationcharacteristicsanddierentiationotwopairsoisomericdiglycosylfavonoidsusingUHPLCESI-Q-ToF-MSMSinnegativeionmode.Ourresultsprovideinor-mativeanduseulMSMSdataandaccurateinormationortherapididenticationosimilartypesofavonoidsinplantormedicinalextracts.MaterialsandmethodsMaterialsandsamplesNaringinglycosylatedwithrhamnosyl-a12-glucosylnarirutinglycosylatedwithrhamnosyl-a16-glucosylneohesperidinglycosylatedwithrhamnosyl-a12-glucosylandhesperidinglycosylatedwithrhamnosyl-a16-glucosylwereobtainedromShanghaiTautoBiotechShanghaiChina.HPLC-grademethanolacetonitrileormicacidandammoniumormatewereobtainedromSigmaStLouisMOUSA.Ultrapurewaterwasusedinallexperiments.DetailsotheisomericdiglycosylfavonoidsarelistedinTable1.Allstandardsweredissolvedinmethanoltoanalconcentrationo20pmolL1oranalysis.InstrumentalanalysisSampleswereanalyzedusinganAgilentAgilentTechnologiesSingaporeUHPLC1290systemcoupledtoaQ-ToFMSMS6540systemAgilentTechnologiesSingaporeequippedwithaJetStreamtechnologyESIionsource.Forchromatographicanalysisa2.1mm100mmAgilentZorbaxC18columnwith1.8mparticlesizewasused.ForthenegativeionmodemobilephaseAcontained0.1vvormicacidinultrapurewaterandmobilephaseBcontained0.1vvormicacidinNameMolecularormulaMHAglyconeR1R2R3NaringinC27H32O14579.1719OR3R2OOHOR1Rhamnosyl-a12-glucosylHOHNarirutinC27H32O14579.1719Rhamnosyl-a16-glucosylHOHNeohesperidinC28H34O15609.1806Rhamnosyl-a12-glucosylOHOCH3HesperidinC28H34O15609.1806Rhamnosyl-a16-glucosylOHOCH3Table1.Structureoselectedisomericdiglycosylfavonoids. X.Guoetal.Eur.J.MassSpectrom.194955201351methanol.Theelutionsolventcomprised10Aand90Batafowrateo200Lmin1.FornegativeionmodetheoptimizedESIsourceconditionswereasollowsgastemperature350Cdryinggasfow8Lmin1nebulizer35psigsheathgastemperature350Csheathgasfow11Lmin1VCap3500Vnozzlevoltageexpt1000Vragmentor300Vskimmer65VOCT1RFVpp750Vandcollisionenergy10eV15eV20eV25eVand30eV.ResultsanddiscussionTheragmentationreactionsotheisomericdiglycosyllavonoidswereanalyzedonthebasisotheirMSMSspectra.TheionnomenclatureortheprecursorionMHothediglycosyllavonoidisomerswasbasedontheionnomenclaturepreviouslyestablishedorlavonoidglyco-sides131417andisprovidedinScheme1.WeusedESI-Q-ToF-MSMSinnegativeionmodetoanalyzetwopairsoisomericdiglycosyllavonoidsnaringinnarirutinandneohesperidinhesperidin.ThemassspectraMSMSotheproductionsotheprecursorionMHoreachisomerwereobtainedateachovecollisionenergies10eV15eV20eV25eVand30eVFigures1and2.DierentiationomassspectraMSMSotheproductionsotheprecursorionMHoisomericdiglycosylfavonoidsFigure1showsthattherelativeintensityotheprecursorionsMHonaringinandnarirutinvariedsignicantlydependingonthecollisionenergy.Therelativeintensitywas100atcollisionenergieso10eVand15eVFigure1A1A2B1andB2.Atcollisionenergieso20eVand25eVtherela-tiveintensityotheprecursorionMHonaringinwasalso100butornarirutinitwas35.09and9.30respectivelyFigure1C1C2D1andD2.Atacollisionenergyo30eVtherelativeintensityotheprecursorionMHonaringinwassignicantlyhigherthanthatonarirutinFigure1E1andE2.ThustheresultsdemonstratedgreaterstructuralstabilityotheprecursorionMHonaringinrelativetothatonarirutin.Figure1showsthattherelativeintensityotheproductionY0otheprecursorionMHdieredsignicantlybetweennaringinandnarirutin.Atcollisionenergieso10eV15eV20eVand25eVtherelativeintensitiesotheproductionsY0onaringinwereclearlylowerthanthoseonarirutinFigure1A1A2B1B2C1C2D1andD2.Therewasanegativecorrela-tionbetweentherelativeintensitiesotheprecursorionsMHandtheproductionY0.TherelativeintensityoY0was100andtherelativeintensityoprecursorionsMHromnaringinwas52.92atacollisionenergyo30eVFigure1E1whiletherelativeintensityoY0was100andtherelativeintensityoprecursorionsMHromnarirutinwas35.09atacollisionenergyo20eVFigure1C2.TheseresultsshowthatinnegativeionmodetheY0cleavagemodedominatedandthatthestructuralstabilityotheprecursorionMHonaringinwasgreaterthanthatonarirutin.Thisisbecausenaringinwasglycosylatedwithrhamnosyl-a12-glucosylandnarirutinwasglycosylatedwithrhamnosyl-a16-glucosyl.Figure1alsoshowsthattherelativeintensityotheproductionmz459otheprecursorionMHwassigniicantlyhigherornaringinthanornarirutin.Fornaringintheproduction459wasproducedrom02X0Hand13AHwhileornarirutinitwasproducedonlyrom02X0Hwhichmayexplainthedierenceinrelativeintensity.Figure2showsthatthechangesintherelativeintensitiesotheproductionsY0andtheprecursorionsMHorneohesperidinandhesperidinandthenegativecorrelationsbetweenY0andMHweresimilartothoseornaringinandnarirutin.TheresultsalsoshowthatY0wasthemaincleavagemodeandthatinnegativeionmodethestructuralstabilityotheprecursorionMHoneohesperidinwasgreaterthanthatohesperidin.Thiswasbecauseneohesperidinwasglycosylatedwithrhamnosyl-a12-glucosylandhesperidinOOHOHOHCH3OOOHOR3OOHOHOHOR2Z1Z0Y0Y1B2B10123413B13A02X002A1012345012345C1C202A202X125A225X025X125A1Z1Z0Y0Y1B2B101234OOOHOR3OOHOHOOHOOHOHOHCH3R2C2C101234501234513B13A02X002A225A225X002A102X125X125A1Scheme1.Ionnomenclatureusedorisomericdiglycosylfavonoids. 52FragmentationCharacterizationandDifferentiationofIsomericDiglycosylFlavonoidswasglycosylatedwithrhamnosyl-a16-glucosyl.Therela-tiveintensityoftheproductionmz489oftheprecursorionMHofneohesperidinwasgreaterthanthatofhesperidin.Forneohesperidinbutnothesperidintheproduction489wasproducedfrom02X0H.Thisprobablyexplainswhytheproductionmz489oftheprecursorionMHofhesperidinwasnotdetectedFigure2A2B2C2D2orhadarelativeintensityofonly0.03Figure2E2.Figure1.ProductionmassspectraoftheprecursorionMHofnaringinandnarirutin. X.Guoetal.Eur.J.MassSpectrom.194955201353ConclusionInthisstudyUHPLCESI-Q-ToF-MSMSwasusedtoiden-tiytwopairsoisomericdiglycosyllavonoidsandthecharacteristicdiagnosticionswereidentied.AnoverviewothecharacteristicdiagnosticionsordiscriminatingisomericdiglycosylfavonoidsisprovidedinFigure3.ThetandemmassspectraotheproductionsotheprecursorionMHotheisomericdiglycosylfavonoidsshowedthatthediagnosticionsweretheprecursorionMHandtheproductionsY0and02X0H.TherelativeintensitiesotheprecursorionsMHandtheproductions02X0HothediglycosylfavonoidsglycosylatedwithFigure2.ProductionmassspectraoftheprecursorionMHofneohesperidinandhesperidin. 54FragmentationCharacterizationandDierentiationoIsomericDiglycosylFlavonoidsrhamnosyl-a12-glucosylnaringinandneohesperidinwerehigherthanthoseotheisomericfavonoidsglycosylatedwithrhamnosyl-a16-glucosylnarirutinandhesperidin.Therewerenegativecorrelationsbetweentherelativeinten-sitiesotheprecursorionsMHandtherelativeintensityotheproductionsY0andY0wasthemaincleavagemodeinnegativeionmode.TherelativeintensitiesotheproductionsY0romthediglycosyllavonoidsglycosylatedwithrhamnosyl-a12-glucosylnaringinandneohesperidinweresignicantlylowerthanthoseotheisomericfavonoidsglycosylatedwithrhamnosyl-a16-glucosylnarirutinandhesperidin.AcknowledgmentsThisresearchwassupportedbytheStateForestryAdministrationP.R.China948ProjectNo.2013-4-07.Reerences1.I.ErlundReviewothefavonoidsquercetinhesperetinandnaringenin.DietarysourcesbioactivitiesbioavailabilityandepidemiologyNutr.Res.248512004.doi10.1016j.nutres.2004.07.0052.X.F.GuoY.D.YueZ.F.MengF.TangJ.WangandX.YaoAntioxidantpropertiesomajorfavonoidsandsubractionsotheextractoPhyllostachyspubescensleavesJ.FoodBiochem.372013.doi10.1111j.1745-4514.2012.00655.x3.B.HalliwellJ.RaterandA.JennerHealthpromotionbyfavonoidstocopherolstocotrienolsandotherphenolsdirectorindirecteectsAntioxidantornotAm.J.Clin.Nutr.81268S2005.4.C.ManachG.WilliamsonC.MorandA.ScalbertandC.RemesyBioavailabilityandbioecacyopolyphenolsinhumans.I.Reviewo97bioavailabilitystudiesAm.J.Clin.Nutr.81230S242S2005.5.K.RobardsandM.AntolovichAnalyticalchemistryoruitbiofavonoidsareviewAnalyst12211R1997.doi10.1039A606499J6.H.J.JeongY.B.RyuS.J.ParkJ.H.KimH.J.KwonJ.H.KimK.H.ParkM.C.RhoandW.S.LeeNeuraminidaseinhibitoryactivitiesofavonolsisolatedromRhodiolarosearootsandtheirinvitroanti-infuenzaviralactivitiesBioorg.Med.Chem.1768162009.doi10.1016j.bmc.2009.08.0367.C.D.KayL.HooperP.A.KroonE.B.RimmandA.CassidyRelativeimpactofavonoidcompositiondoseandstructureonvascularunctionAsystematicrevieworandomisedcontrolledtrialsofavonoid-richoodproductsMol.Nutr.FoodRes.5616052012.doi10.1002mnr.2012003638.R.GautamandS.M.JachakRecentdevelopmentsinanti-infammatorynaturalproductsMed.Res.Rev.297672009.doi10.1002med.201569.I.SnchezF.Gmez-GaribayJ.TaboadaandB.H.RuizAntiviraleectofavonoidsontheDenguevirusPhytother.Res.14892000.doi10.1002SICI1099-157320000310.X.ZhengP.ShiY.ChengandH.QuRapidanalysisoaChineseherbalprescriptionbyliquidchromatographytime-o-fighttandemmassspectrometryJ.Chromatogr.A12061402008.doi10.1016j.chroma.2008.08.038MHPrecursorionYesNo1HigherrelativeintensityofMHQ-TOF-MSMS-ESIFrag300VCID1030eVIsomericBIsomericAIsomericAIsomericdiglycosylfavonoidglycosylatedwithrhamnosyl-12-glucosylIsomericBIsomericdiglycosylfavonoidglycosylatedwithrhamnosyl-16-glucosyl2LowerrelativeintensityofY03Higherrelativeintensityof02X0-H-Figure3.Guidelinesordiscriminatingamongdiglycosylfavonoidisomers. 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57ISSN1469-0667IMPublicationsLLP2013doi10.1255ejms.1213AllrightsreservedEUROPEANJOURNALOFMASSSPECTROMETRYWereportedinapreviousshortcommunicationonthedetectiononewlinearpolyaminesandpolyaminederivativesinthevenomsothespidersAgelenopsisapertaHololenacurtaandParacoelotesbirulaiTable1.1Thearticleocusedprincipallyonthechemo-diversityospidervenomsomittingthedetailsotheanalyticalproceduresanddeductionsthatledtothestructuralelucidationsothecompounds.Inthisreportwewouldliketoclosethisgapbyshowingtheapproachweusedtouncoverthestructuresothepolyaminederivatives118primarilyusingthecharacteristicsoknowntandemmassspectrometryMSMSragmentationpatterns.Inadditionwepresenttwonewacylpolyaminetoxins19and20oundinP.birulai.PolyaminesarewidelyspreadthroughoutnatureandtheirMSanalysisparallelstheevolutionothemethod.Moresophisticatedapplicationsstartedinthemid1970swhenBiemannetal.usedtheelectronimpactEIragmenta-tionbehavioroderivatizedpolyaminoalcoholsorpeptidesequencing.24WiththeadventomilderionizationmethodsGrossetal.wereabletoanalyzerespectivepolyaminoalcoholsdirectlybyastatombombardmentFABMSMS.5Theanal-ysisopolyaminespidertoxinsbymeansoMScanlookbacktotheearly1990swhenQuistadetal.threedecadesaterthefrstreportontheoccurrenceopolyaminederivativesinspidervenoms6usedMSMSorthecharacterizationotheAtemplateapproachorthecharacterizationolinearpolyaminesandderivativesinspidervenomManuelTzourosSergeChesnovLaurentBiglerandSteanBienzInstituteoOrganicChemistryUniversityoZurichWinterthurerstrasse190CH-8057ZurichSwitzerland.E-mailsbienzoci.uzh.chAcombinationohigh-perormanceliquidchromatographyHPLCandatmospheric-pressurechemicalionizationmassspectrometryAPCI-MSandAPCI-MSMSwasusedtodetectandcharacterizelinearpolyaminederivativesinthevenomothespidersAgelenopsisapertaHololenacurtaandParacoelotesbirulai.Thecompoundswereidentifedwithatemplateapproachbywhichthecollision-induceddissociationCIDspectraoknowncompoundsaredirectlycomparedandcorrelatedwiththoseotheanalytes.Toacilitatetheperceptionothespectraandtherecognitionothestructuraleaturesotheanalytesanionnomenclaturecloselyleanedontheacceptednomenclatureorragmentionsopeptidesornucleicacidsisintroduced.ThestructureidentifcationopolyaminederivativesbydirectcorrelationoMSspectraispossiblebecausesuchcompoundsshowverydistinctiveragmentationbehavior.Oparticularrelevanceistheactthatthesignalpatternsthatareobservedwithanalytespossessingdierentpolyaminebackbonesarenotonlydistinctwithregardtomassdistributionsbutalsowithregardtorelativesignalintensitiesresultinginfngerprint-likesignalpatterns.Thedirectcorrelationothesepatternsmorethanthecorrelationotheiondistributionswasoundtobeokeysignifcance.Withthisthenewapproachisundamentallydierentromthesequencingopeptidesornucleicacidswhicharelargelybasedonmassdistributions.ThemethodismoreefcientandmorereliablethanthedenovointerpretationotheMSdataanditevenallowstheiden-tifcationopolyamineportionsincompoundsthatareanalyzedwithinmixtures.KeywordsspidertoxinstriplequadrupoleMStemplatepolyamineacetylguanidylIntroductionM.Tzourosetal.Eur.J.MassSpectrom.1957692013Received22February2013nAccepted12March2013nPublication13March2013 58LinearPolyamineDerivativesinSpiderVenomSourceCompoundabtRmingradientcMHmzStructuresExamplesA.apertaH.curtaPA34315.45.5a203PA33425.45.5a203H2NHNHNHNNHPA33343NH233343PA334335.45.5a260PA333445.45.5a260PA3343355.45.5a317PA3334365.45.5a317PA3333475.45.5a317A.apertaH.curtaPA3OH4385.45.5a219H2NNHNHNNHPA3OH3343NH233343OHPA3OH3495.45.5a219PA3OH343105.45.5a276PA3OH334115.45.5a276PA3OH3343125.45.5a333P.birulaiPA3334Gu1312.3a302HNHNHNHNHNAc33334GuNH33334H3CONH2NHPA33334Gu1412.9a359PA3OH334Gu1512.3a318Ac3334Gu1612.9a344Ac33334Gu1712.9a401H.curtaAc33343Ac1812.8a401P.birulaiIndAc3334Gu4-OH-IndAc33334Gu192027.9b25.9b459532NHHNHNHNHNONHIndAc3334GuNH2NH3334HNNHNHNHN4-OH-IndAc33334GuNH33334OHONH2NHNHOHaPAstandsorpolyaminethefguresdesignatethenumbersomethyleneunitsin-betweentheseveralN-atoms.bAbbreviationsorthesubstituentsAcCH3COGuH2NCNHIndAcand4-OHIndAcseeex-amplestructures.cForthegradientsseeExperimental.Table1.PolyaminesandpolyaminederivativesfoundinspiderVenomofAgelenopsisapertaHololenacurtaandParacoelotesbirulai. M.Tzourosetal.Eur.J.MassSpectrom.195769201359mostabundantpolyamintoxinsothespidersAgelenopsisaperta7andHololenacurta.8Inthemeantimemanydierentspiderspecieshavebeeninvestigatedbyseveralgroupsandtheanalyticalprocedureshavebeenwellrened.920Todaysanalyticalsetupsochoicearearraysohigh-perormanceliquidchromatographyHPLCandelectrosprayoratmos-pheric-pressurechemicalionizationmassspectrometryESI-MSorAPCI-MScombinedwithultravioletdiodearraydetectionUV-DAD.ThesearrangementsnotonlyallowtheseparationanddetectionolowlyabundantpolyaminetoxinswithincomplexmixtureswhichisthegeneralchallengetobeconrontedwithwhenworkingwithspidervenomsbutinmostcasesalsothedirectstructuraldeductionothesamplemoleculesbyMSMSormulti-stagemassspectrometryMSn.Wehavecontributedtothetopicbyinvestigatingthetoxinsoseveralspiderspeciesundertakinginparticularin-depthstudiesotheragmentationateosuchcompoundsuponcollision-induceddissociationCID.162126Thesestudieshaveresultedinacollectionodatathatnowallowthestraight-orwardstructuralelucidationounknownpolyaminederiva-tivesbytheapplicationosomerathersimpleragmentationrulesorbydirectcomparisonoanalyteMSMSwithspectraltemplatesobtainedromreerencecompounds.ExperimentalMaterialReerencePA3343andPA3334werepreparedbysolid-phasesynthesisanalogoustoaprocedurereportedearlier.23SamplesolyophilizedspidervenomromA.apertaH.curtaandP.birulaiwerepurchasedromFaunaLaboratoriesLtdAlmatyKazakhstan.Crudevenom0.5mgwasdissolvedin50Loa32volvolmixtureoacetonitrileHPLCgradeScharlauBarcelonaSpainandwaterpuriedbyMilli-QRGMilliporeMilordMAUSAcontaining0.1trifuoroaceticacidTFAFlukaBuchsSwitzerland.TheclearstocksolutionsovenomweredirectlyusedorHPLC-MSorstoredat20Cpriortouse.LiquidchromatographyChromatographicseparationswereperormedwitha626-LCsystemequippedwitha600ScontrollerWatersCorporationMilordMAUSAanda7725irotaryvalvewitha5LinjectionloopRheodyneCotatiCAUSA.AMillenniumChromatographyManager2010v2.15wasusedorthecontrolotheinstrument.TheanalyteswereseparatedonanUptisphereUP3HDO20QSC18reversed-phasecolumn2004.6mmi.d.particlesize3mInterchimMontluonFranceatafowrateo0.5mLmin1withsolventsA0.1TFAinwaterandB0.1TFAinacetonitrileandoneotheollowinggradientsarom0to10oBover5minthento15oBover10minbrom0to10oBover5minthento33oBover45min.Attheendoeachgradientthecolumnwaswashedwith80oBor5minollowedbyequilibrationwith100oA.TheretentiontimetRaswellasthegradientusedoreachidentiedcompound120isgiveninTable1.MassspectrometryTheAPCI-MSandAPCI-MSMSexperimentswereperormedonaFinniganTSQ700triple-quadrupoleinstrumentequippedwithanatmospheric-pressurechemicalionizationsourceAPCIFinniganMATSanJoseCAUSAandcontrolledbytheICISsotwarev8.3.TheAPCIoperatingconditionsinposi-tivemodewerevaporizertemperature450Ccoronavoltage5kVheatedcapillarytemperature250CsheathgasN2withaninletpressureo40psielectronmultiplier1kVconver-siondynode15kV.Theresolutionwasadjustedto0.7uathalpeakheightFWHM.TogetrepresentativeMSspectra16scanswereaveraged.TheHPLC-MSMSmeasurementswereperormedbyreplicateinjectionothesamplewithArasacollisiongasatarelativepressureo2.53.3mTorrandwiththeelectronmultipliersetat1.5kV.Precursorioncandidatesorcollision-induceddissociationCIDwereselectedusingtheinitialHPLC-MSrunsettingtherstquad-rupoleQ1attherespectivemzvaluesandscanningthethirdquadrupoleQ3overatypical50550mzrange.TheCIDosetvoltageCowasadjustedto20VorallcompoundsexceptorPA33334Gu14Ac3334Gu16andAc33343Ac18to25VandorIndAc3334Gu19and4-OH-IndAc33334Gu20to27V.MSMSscanswereaveragedovertherespectivechromatographicpeaks.ResultsanddiscussionFragmentationbehaviorofacylpolyaminederivativesuponCIDTheuseoragmentationrulesorospectraltemplatestoidentiystructuralunitsinpolyaminederivativesispossibleonlybecausethecompoundsollowrathercommonandcharacteristicragmentationreactionsuponCID.Theionpatternsobservedromsuchcompoundsarenotonlytypicalwithregardtomassdierencesbuttoagreatextentalsowithregardtorelativeionintensities.Thusagivenpoly-aminerameworkusuallyconsistingoadiaminobutaneunitlesscommonlyoadiaminopentaneunitandoneorseveraldiaminopropaneunitsgivesrisetoauniqueragmentationpatternthatcanbeconsideredasadiagnosticngerprintothemolecularportionunderinvestigation.ForinstancetheourisomericspidertoxinsIndAc3334IndAc3343IndAc3433andIndAc4333AG416aAG416AG416candAG416bdiscussedearlier2324seealsoFigure1SintheSupplementaryMaterialhavenallybeenidentiedonthebasisosuchuniquepatterns.Thecompoundsdiersolelyinthelocationothediaminobutanemoietywithinthepentaminebackbonesbuttheirspectraarequitedierent.TheourMSMSnotonlyshowsomediagnosticsignalsoreachindividualsamplecompoundbuteachothemalsoleavesitsownchar-acteristicoverallimpression.Onlythiscombinationinallyallowedtheunambiguousidenticationotheourcompounds 60LinearPolyamineDerivativesinSpiderVenomasnaturaltoxinswithinthecomplexmixtureofthenativespidervenom.Thepredominantfragmentationreactionobservedwithpolyaminebackbonesistheintramolecularnucleophilicsubstitutionofaprotonatedaminoorguanidinogroupasrevealedbyseveralstudiesperformedbyus2124andbyothers2728withsyntheticreferencecompoundsisotopelabe-lingandMOcalculationsScheme1.ThesereactionsleadinthecaseofsimplepolyamineframeworksofthetypeAtotheformationofachargedcyclicstructuresuchasBbylossofanunchargedaminesuchasC.Sincethenucleophilicsubstitutioncanoccurateachdiaminoalkanemoietyofthepolyaminebackboneandforagivendiaminoalkanemoietylikewisefromthelefttorightfull-linedarrowsorfromtherighttoleftdashed-linedarrowsthechainofthepoly-aminemoietycanvirtuallybewalkedalongstartingfrombothsidesofthemolecule.Similartothenomenclatureusedinpeptideornucleicacidfragmentationswithoutmeaningtoimplysimilarstructuresorsimilarfragmentationreac-tionsionseriesofthetypeastartingfromtheacylatedendofthemoleculeandzstartingfromthebasicaminoendofthemoleculecanbeproposed.InadditiontotheaandztypeionsalsoionsofthetypeanH2OoftypebionsandznNH3ofthetypeyionsaredetected.ThebnionseitherderivefromtherespectiveanionsbylossofwaterorfromtheMHH2Oionionbwbyintramolecularsubstitutionreactions.TheynionsresultfromtheexpulsionoftheterminalNH2groupinformofNH3.Inadditiontotheabandzytypeionsalsoionsoftruncatedmolecularionsareobservedtseries.TheseareformedbyneutrallossofznHportionspossiblythroughHtransferinintermediaryproton-boundcomplexes.24ThesetruncatedionsleadtozandyionsbyintramolecularsubstitutionsasdescribedfortheMHprecursorions.AsanexamplethecompleteinterpretationoftheCIDspectrumofIndAc333423isshowninFigure1.NHRNH2RnNnRHH2NRNHHNHNHNHNONH2xa4a3a2a1z1z2z3HABCScheme1.FragmentationofpolyaminebackbonesbyintramolecularnucleophilicsubstitutionsassumeprotonationoftheleavingamineN-atomintheprecursorion.10080604020417399289215186129112IndAc3334AG416a3463292727298382311169115400MHa4a3a2a1b4bb3z1z2z3z2y2y2y3t1t2100200300400mzRelativeIntensity84NHHNHNHNHNONH2a4a3a2a1z1z2z3b4b3y2y32Ht12Ht2MHHH2OH2ObH2ONH3NH3z2y2NH3HFigure1.FragmentationschemeforIndAc3334AG416aandrespectiveCIDspectrum. M.Tzourosetal.Eur.J.MassSpectrom.195769201361AscanberecognizedromthespectruminFigure1thesuccessionothesignalsintheseveralionseriesgivesdirectevidenceortheconstructionothepolyaminebackboneothesamplemolecule.Howeverstructuralelucidationsbasedontheoccurrenceorabsenceotheseragmentsaloneareusuallynotpossibleorpolyaminespidertoxinsromnaturalsources.Thisisortworeasons1someotheapparentlydiagnosticsignalscanariseromalternativeragmenta-tionpathwaysorexampletheionsothezandyseriesatmz11598couldbemisreadasconclusiveoraterminalPA33portioninAG416a24and2spidertoxinsotenhavetobeanalyzedwithinmixturesostructurallyverysimilarcompounds2223wherethesignaldistributionsderivedromsuperimposedspectranolongerunambiguouslycharacterizetheunderlyingsamplecompounds.FortunatelytheparticularcharacteristicsanddiagnosticdierencesotheCIDspectraopolyaminederivativesarenotonlyduetothedierentragmentseriesbutalsoduetocharacteristicrelativeionintensitiesthesignalpatterns.Thebasisorthesubstrate-specifciondistributionsliesintheactthatragmentationaroundthediaminobutanemoietyothemoleculesisstronglypreerred.SubstitutionreactionsinitiatedbyN-atomsattachedtoaC4-unitproceedthroughavoredive-memberedcyclictransitionstructuresandareaccordinglypreerred.Therelatedionsignalsarethusobservedwithhigherintensitiesinsomecasestheydominatethespectrainawaythatexpectedsignalsaresuppressedandnotdetectedatall.23N-hydroxylatedanalogsolinearpolyaminesbehaveverysimilarlytothecompoundsdiscussedabove.Thesecompoundsexhibitanalogousfngerprint-likepatternswiththedistinctdierencethatlossowaterormationoionsothetypebisusuallynotobserved.AsanexampletheragmentationschemeandtheCIDspectrumoIndAc3OH3343AG48922orHO4897isoutlinedinFigure2SintheSupplementaryMaterial.InsummaryitcanbestatedthatthephenotypesotheCIDspectraobtainedromcompoundswithpolyaminebackbonesarecharacteristicenoughthatspectraowell-characterizedreerencecompoundscanbeusedasingerprint-liketemplatestoidentiyrelatedstructures.Ratherthaniondistributionswhichcanleadtomisinterpretationstheoverallappearanceothespectraisimportant.ThisdistinguishesthetemplateapproachromclassicalMS-basedsequencingasitisappliedinpeptideandnucleicacidanalysis.Identifcationonon-acylatedpolyaminederivativesusingMStemplatesTheproblemounsaestructureidentifcationonthebasisotheobservedragmentmassesaloneisevenmorepronouncedwhenterminallynon-derivatizedpolyaminederivativesareanalyzed.Inthesecasesacyllabelsthatallowtodistinguishtheright-sideromthelet-sideendsothemoleculesaremissing.Thusthesametypesoragmentationswilloccurstartingrombothendsotheprecursorionsgivingriseonlytozyandttyperagmentions.TheseionscanbelabeledwiththesuperscriptsLandRrespectivelytodistinguishbetweenthesitesothemoleculetheyariseromseeinsertsinFigure2.Anon-acyl-terminatedpolyaminethereoreshowsragmentationeaturesthatarerelatedtotwotypesoacylatedpolyamines.ForinstancePA3343isspectrallyrelatedtobothAcyl3343andAcyl3433IisomericpolyamineshavetobedierentiatedorinstancePA33433andPA33344thebis-directionalN-terminusragmentationothecompoundsrepresentsaproblemseeinsertsinFigure2.Inactthereisnoionsignaltobeimag-inedorPA3343thatcouldberegardedasuniquelyassociatedtothecompoundbecausetheisomericPA3334wouldallowtheormationoalltheionsthatcanbeexpectedorPA3343aswell.AnintrinsicdiagnosticsignalhowevercanbeexpectedorPA3334.Onlythiscompoundcanleadtoaragmentionwiththemass172uLz3becauseonlythiscompoundhasaterminaldiaminobutanemoietythatcanbeexpelled.ToexcludethepresenceoPA3343onthebasisoanobservedragmentionwiththemass172uhoweverisnotvalidanditisalsonotallowedtodeducePA3343asthestructureoracompoundjustbecausenosignalwasobservedatmz172.InthefrstcasethesamplemightbeamixtureoPA3334andPA3343andinthelattercasetheallegeddiagnosticragmentionmightnotbeormedromtheparticularpolyaminederiva-tivedespiteitsterminaldiaminobutanemoiety.TheragmentationbehaviorothetwocompoundsPA3343spectrumainFigure2andPA3334spectrumbinFigure2howeveraresodistinctivelydierentthattheirspectracombiningsignallocationandsignalintensitiescanbeusedastemplatesortheidentifcationounkowns.ItcanreadilyberecognizedthatthechromatographicractionoanaturalvenomsampleoAgelenopsisapertagivingrisetospectrumcinFigure2containsbothpolyamines.Oparticularsignif-cancearethesignalsoionsthatariseromragmentationaroundthePA4unitothemolecules.Forclaritythediag-nosticpatternsorthetwocompoundsarehighlightedinthespectrainblueandred.WiththeMStemplatesoPA3343andPA3334athandandtheknowledgeothediagnosticznandtnsignalstobeexpectedoreachthethreeisomerichexaminesPA33433PA33343andPA33334compounds57wereidentifedasco-elutingcomponentsinchromatographicractionsotheH.curtaandA.apertavenomsseeTable1.TheMStemplatesoPA3343andPA3334cannotonlybeusedtoidentiyhomologeouslinearN-unsubstitutedpoly-aminesbutalsotocharacterizeN-hydroxylatedpolyamineanalogs.Figure3showsthatthevenomoA.apertacontainsinadditiontothetwopolyaminesPA3343andPA33343and4co-elutinginHPLCspectrumbtheN-hydroxylatedderivativesPA3OH343andPA3OH33410and11alsoco-elutinginHPLCspectrumaaswell.Anumberosignalscorrespondingtoragmentionsderivingromthecommonnon-hydroxylatedportionsothemoleculesareoundatthesamemzvaluesinbothspectrawhileothersignalsaredetectedinthetwoMSwithmassshitso16uspectralportionsinred.ThelatternotonlyindicatesoxidationbutalsoallowslocalizationotheOHgroupat 62LinearPolyamineDerivativesinSpiderVenomN2ofthepolyamine.ThesignalpatternslabeledinblueandredarecharacteristicofthePA3343PA3OH343andPA3334PA3OH334frameworksrespectively.EventhoughthepresenceofananalytecannotbeexcludedunambigouslyonthebasisofmissingsignalsinaspectrumnoindicationfortheexistenceofPA3OH433orPA4OH333wasfoundinspectrumaorinanyspectrumoftheHPLC-MSrun.OnlymarginalifanysignalintensitywasobservedforLz2Ly2orRz3ionsofPA3OH433atmz145mz128ormz186orofRz3andRy3ionsofPA4OH333atmz172andmz155fragmentionsthatwouldbeexpectedtobeformedabundantlyfromthetwocompounds.TheCIDspectrumofthevenomfractionthatconsistsofthethreeisomerichexaminesPA33433PA33343andPA3333457allowedtheidentificationoftheN-hydroxylatedderivativePA3OH334312.AlignmentofthespectracanddasshowninFigure3revealsthatsolelyoneisomericformofthehexaminederivativeiscontainedinthechromatographicfractionofthevenom.InspectrumconlyonecorrelationtospectrumdcanberecognizedthecorrelationtothediagnosticionpatternofPA33343shiftedby16massunitstoaccountfortheadditionaloxygenatomhighlightedinred.ScrutinizingthecompletespectrumcrevealedthatallsignalsexceptforoneareconsistentwiththestructurePA3OH334oftheanalyte.TheonlysignalthatcannotbeexplainedwithstructurePA3OH334isfoundatmz172.ThissignalwouldcorrespondtoanionthatcouldarisefromacompoundorafragmentwithaterminalPA333Figure2.CIDspectraoasyntheticPA33433bPA33344andcaractionoA.apertavenomthatcontainsamixtureothetwocompounds.23TheragmentationschemesorPA3343andPA3334areinsertedandthediagnosticsignalsorthetwocompoundsarehighlightedinblueandred.Acolourversionothisfgurecanbeoundontheweb. M.Tzourosetal.Eur.J.MassSpectrom.195769201363243259MH276mz10058728498112115129186260MH169172189100114131202188205RelativeIntensityRelativeIntensity16uPA3OH343PA3OH334abPA3343PA3334587284981121291858060402040806020588498112115129246203317MH186189300114131188205259MH333mz16u2432437272169172PA3OH3343PA33433PA33343PA33334588498112129169172100100RelativeIntensityRelativeIntensitycd8060402040806020xxxxFigure3.ComparisonsoCIDspectraoseveralractionsoA.apertavenom.SpectrumbobtainedromaractionthatcontainsPA33433andPA33344wasusedasthetemplatetoidentiytheconstituentsPA3OH34310andPA3OH33411otheractionprovidingspectruma.SpectrumcwasobtainedromaractionthatcontainssolelyPA3OH334312asrevealedbythecomparisonwithspectrumdmeasuredwitharactionthatcontainsthethreehexaminesPA33433PA33343andPA3333457.Thediagnosticsignalpatternsorrelatedpolyaminebackbonesarehighlightedinmatchingcolors.Acolourversionothisfgureisavailableonourwebsite. 64LinearPolyamineDerivativesinSpiderVenomportion.Howevernootherindicationorthepresenceosuchastructuralunitisoundinthespectrum.ThespectralcomparisonsshowninFigure3areexamplesthatshowthattheMStemplateapproachisnotonlysuitedtoidentiyingananalytebyapositivematchbutalsotoexcludepotentialstructuresbymissingcorrelations.CorrelationoreepolyamineswithacylployaminesWealreadyreporteduponanumberoacylatedpolyaminederivativesromthevenomoseveralspiders.162126Thestruc-tureelucidationsothesecompoundswerecareullyperormedbyMSMSanalysisstudyotheragmentationpathsandinsomecasescomparisonwithullycharacterizedsyntheticreerencecompounds.ComparisonotheCID-MSotheseacylpolyamineswiththoseotheparentpolyaminesrevealedthattheragmentationpatternsorthesharedstructuralunitsareconservedinthiscaseaswell.Thusthetemplateapproachortheidentifcationopolyamineportionsalsocanbeappliedwithspectraoacylpolyaminesasthereerences.Thisisdemonstratedwithtworepresentativeexamples.InFigure4thespectraothenaturalmixturesoPA3OH343andPA3OH33410and11spectrumaandoIndAc3OH343andIndAc3OH334AG432andAG432gspectrumb22areshown.Theanalogyothesignalpatternsinthetwospectrawiththeportionscorrespondingtosignalsderivingromletportionsotheanalytesshitedby157uduetothepresenceorthelackotheIndAcmoietyisquiteevidentandbythisalsothepresenceothesamepolyaminebackbonesinthetwosamples.aThusthetemplatespectrumotheIndAcderivativesconfrmstheassignmentoPA3OH343andPA3OH334astheanalytesprovidingspectrumaasdeducedwiththetemplatespectrumothenon-oxidizedpolyaminesseeFigure4.ThesameanalogyosignalpatternsandspectralshitsasobservedorthepentaminederivativeswasobservedorthehexaminederivativesPA3OH334312andIndAc3OH3343AG489sampleormP.birulai21showninFigure3SintheSupplementaryMaterial.IdentifcationoacetylatedandguanidinopolyaminederivativesSincetheacetylandtheaminoiminomethylgroupssharethesamenominalmassitwasexpectedthattheidentif-mzPA3OH343andPA3OH334IndAc3OH343AG432fandIndAc3OH334AG432g100100RelativeIntensityRelativeIntensityab80604020408060201121291121295858728484987298114115131202202215285359188345205259MH276288362416433MH185148305157uFigure4.ComparisonoCIDspectraotworactionsoA.apertavenom.TheconstituentsPA3OH34310andPA3OH33411otheractionprovidingspectrumawereidentifedwiththespectraltemplatebderivingromaractionthatcontainsIndAc3OH343andIndAc3OH334AG432andAG432g.22Thediagnosticsignalpatternsorthetwopolyaminebackbonesarehighlightedinmatchingcolors.Acolourversionothisfgureisavailableonourwebsite.aTheonlyevidentdierencebetweenthetwospectraarethedierentintensitiesothesignalsatmz58spectrumaandmz215spectrumbrespectivelyrelativetotherestothespectra.ThesesignalshowevercorrespondtotheLz1anda1ragmentsotwocompoundswhichcannotbecompareddirectly. M.Tzourosetal.Eur.J.MassSpectrom.195769201365cationanddierentiationothesetwogroupsmightrepre-sentaproblemwhenusinglowresolutionMS.Howeverthisisnotthecase.Wehavealreadylearnedwiththestruc-turalelucidationo4-OH-IndAc3OH334GuPB49021thatguanidinoderivativesshowadistinctragmentationbehavior.Therespectiveionchemistrywasinvestigatedinmoredetailwithargininederivatives2728StepwiselossotwomoleculesoNH3ormationocwMH17anddwMH1717lossocarbodiimidetwMH42andlossoguanidinea4MH59romtheprecursorionsistypicalorsuchcompounds.Alsocharacteristicistheormationotheionsfeandz1withmasseso112u154uand114u.TheionsandtherespectiveragmentationpathsareshowninScheme2thecharacteristicsignalpatternuponCIDareoundinspectrumaoFigure5.Incontrasttotheguanidinocompoundstheacetylatedpolyaminederivativesshownospecialbehavior.CIDleadstotheusualprominentormationoaandbtyperagmentsappearinginpairsanddieringinmassby18u.Theacetylmoietyitselisrecognizedwithragmenta1recordedinourcaseortheexclusivelyoundAc3......3Acmoietiesatmz100.Spectrumbothebis-acetylatedderivativeAc33343Ac18inFigure5nicelyillustratesthisbehavior.Theidentiicationotheseveralacetylandguanidinopolyaminederivatives1317wasthenpossibleusingthetemplateapproachdescribedabove.ForinstancespectrumcinFigure5readilyconfrmsthatcompound16Ac3334Gupossessesbothan...34GuandaAc3...terminus.Therespec-tiveionpatternsarehighlightedinredandblue.ThemissingpropyleneunittocompletethestructureothepolyaminebackboneoAc3334Guwasfnallydeducedromthesignalorthea2ionsmz157andromtheoverallmolecularmasso343u.Startingwiththespectrumo4-OH-IndAc3OH334GuPB490astheinitialMS-templatealsothenon-acylatedpentaminederivativePA3OH334Gu15anditsnon-N-hydroxylatedequivalentPA3334Gu13wereeasilyidentifed.WiththespectrumothelattertheidentityothehexaminederivativePA33334Gu14wasascertainedandtheMSpatternsoPA3334GuandPA33334GuwerefnallyalsooundintheMSotheacetylatedderivativesAc3334Gu16andAc33334Gu17.RepresentativelythespectralcomparisonsoNHHNNHNHNONHa4z1MHHOHOHNHNH22HtcNH3dNH3diimine42u4-OH-IndAc3OH334GuPB490e154uguanidine59u114uHNNHNH3NHHNRNNHNHHNRHNH3cNNHNH2HNRNNHNHH2NRNf112uNNHNRdNH3NNHNHe154uScheme2.Typicalfragmentsformedfromguanidinoderivatives2728shownwiththespidertoxinPB490fromP.birulai.21 66LinearPolyamineDerivativesinSpiderVenomAc3334Gu16PA3334Gu13andPA33334Gu14isshowninFigure4SintheSupplementaryMaterial.Itisinterestingtomentionatthispointthatwefoundinallinvestigationsofpolyaminespidertoxinssolelythe...34GuRelativeIntensity361a443237830184231211169e154f11298c474t449d457z1114318100806040mz100200300400500mz100200300400500mz10020030040050020MH491174259174-OH-IndAc3OH334GuPB490RelativeIntensity1008060402098112129115a1100Ra2171La2157La3214Lb3196Ra4285Rb4267MH401b383181818Ac33343AcRelativeIntensityAc3334Gu11211498231256196285302310327326MH344267214211194169154a2157100847210080604020181818abcFigure5.CIDspectraoaPB490romP.bilurai21bAc33343Ac18andcAc3334Gu16withthetypicalsignalpatternsorthe...34GuterminushighlightedinredandtheAc3......3Acterminihighlightedinblue.Acolourversionothisfgureisavailableonourwebsite. M.Tzourosetal.Eur.J.MassSpectrom.195769201367terminusno...33Guor...43Guterminiinthepolyamineback-bone.121Weassumethatthishasabiogeneticreason.Webelievethattheguanidino-terminatedpolyaminebackbonesarebiosynthesizedromagmatinewhichitselarisesromargininebydecarboxylationundertheactionoanargininedecarboxylase.IdentifcationonewguanidinoacylpolyaminespidertoxinsWiththespectraltemplatesoPA3334GuAc3334GuandPA33334GuAc33334GuavailabletwoormerlyunknownspidertoxinsoP.birulaiIndAc3334Gu19and4-OH-IndAc33334Gu20wereidentiiedorcompletestructuresseeTable1.Thespectralcomparisonothepairo4-OH-IndAc33334GuAc33334GuisshownrepresentativelyinFigure6.ItisreadilyrecognizedromthespectrainFigure6thatthereisaclosespectralpropinquity.Inparticularthecompletesetosignalsorthea-andb-typeragmentsareoundthroughoutbothspectra.Alsotheimportantsignalsrelatedtothe...34Guportionareoundinbothspectra.Thusthestructureo4-OH-IndAc33334Gu20asananalytecanberegardedassecured.Howeveraratherbroadsetosignalsinthespectrumo4-OH-IndAc33334GuhasnorelationtothespectrumoAc33334Gu.Thesesignalsmighthavetheirsourceinaco-elutinganalytethatisisobaricto4-OH-IndAc33334Gu.ConclusionWehaveshownwithseveralexamplesthatCIDspectraoknownpolyaminederivativeseveninmixturescanbeusedastemplatesortherapidandreliableidentifcationopolyaminebackbonesounknownanalytes.Sincenotonlysignallocationsbutalsoevenmorecharacteristicallysignaldistributionspatternsarediagnosticoragivenpolyaminemoietythedirectcomparisonospectrabecomesmoreefcientandalsomorereliablethandenovointerpretationothespectra.Thenewlyintroducedionnomenclatureortheragmentationproductsopolyaminederivativescloselyleanedontheacceptednomen-clatureorragmentionsopeptidesornucleicacidsacilitatestheperceptionotheCIDspectraandallowsamoreacilerecognitionostructuraleaturesotheanalytes.ab98a1231a2288y2154z2171b2170b3327b4384b5455a4402a5473Rt490a3345Lt359d498c515Rt141998112114z1112z1114129169169100a1157a2139b2196b3253b4324b5342a5359Rt367d384c271a4288Rt1214a3154y2171z2100100RelativeIntensityRelativeIntensity8060402040806020401MHMH5324-OH-IndAc33334GuAc33334Gum131uFigure6.ComparisonoCIDspectraotworactionsP.birulaivenomcontaining4-OH-IndAc33334Gu20spectrumaandAc33334Gu17spectrumb.Thesignalsoz-andy-typeragmentsthatareretainedinthetwospectraarehighlightedinbluethesignalsothea-b-c-andt-typeionsthatareshitedby131uarehighlightedinred.Acolourversionothisfgureisavailableonourwebsite. 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71ISSN1469-0667IMPublicationsLLP2013doi10.1255ejms.1210AllrightsreservedEUROPEANJOURNALOFMASSSPECTROMETRYLivingcellsusetheredoxpropertiesocopperionsinnumerousphysiologicprocesseswhereasGSHbalanceandcopperhomeostasisareunctionallylinkedandinvolvedincellprolieration.12Thereisaconsiderableamountooxidizedglutathioneinlivingcellswhichisselectivelystashedinvacuoles.3ThereeCuIIionisreducedbyGSHwiththeormationosuperoxideradicalsandtheirreversibleconver-sionoCuIGSHintotheredox-inactiveCuIIGSSGcomplex.45HoweverthereducedglutathioneascorbateandsuperoxideareabletoregenerateCuIGSHromCuIIGSSG.6Glutathionedepletioninneuronsandaberrantcoppermetabolismhavebeenimplicatedinseveralneurodegen-erativedisorders.7AlthoughtheinteractionometalionswithpeptideshasbeenstudiedbyESI-MStoourknowledgelittleinormationontheafnityocopperionsorGSHisavailable.UnderthesecircumstancesthealreadyreportedinliteraturecomplexesCuIGSH2andCuIIGSSGcouldbeatargetinMSstudies.ThereoretheaimothisworkwastoinvestigatetheeectoreecopperionsandalkalineenvironmentontheGSHoxidationandtheormationocopperglutathionecomplexes.ThisstudyshowedthatitispossibletoollowbyMStheequilibriumbetweenreducedandoxidizedormsoGSHandthatbetweenCuIGSH2andCuIIGSSGcomplexes.ExperimentalESI-MSmeasurementswerecarriedoutonaBrukerDaltonicsEsquire3000plusBremenGermanyiontrapmassspec-trometer.Spectrawereacquiredinthe1002000mzrangeinpositivemode.Drygaswithadrytemperatureo300Cwasintroducedatarateo5.0Lmin1.Thecapillaryexitwas197.5Vandskim62.4V.ThepHvaluesoallsolutionswerecheckedwithaHANNAPH211microprocessorpHmeterbeoreandaterthetreatmentwithcopperions.AllchemicalswereanalyticalreagentgradeandallsolutionspreparedwithmilliQgradewaterwithR18.2M.CoppersaltsammoniumbicarbonateandglutathionewerepurchasedromLetterMassspectrometricapproachofhighpH-andcopper-inducedglutathioneoxidationGabiDrochioiuLauraIonCatalinaCiobanuLauraHabasescuandIonelMangalagiuFacultyoChemistryAl.I.CuzaUniversityoIasi11CarolIIasi-700506Romania.E-mailgabidruaic.roTheinteractionbetweencopperionsandg-L-glutamylL-cysteinylglycineglutathioneGSHmoleculesmayleadtotheformationofthephysiologicallyoccurringCuIGSH2andCuIIGSSGcomplexes.SinceglutathionedepletioninneuronsandaberrantcoppermetabolismhavebeenimplicatedinseveralneurodegenerativedisorderswestudiedheretheinteractionofGSHwithcopperionsCu2byelectrosprayionizationiontrapmassspectrometryESI-IT-MS.BesidesweextendedthisapproachtopHinexcessof10byaddingethanolaminetothesolutionbeinginvestigated.AsaresulttheESI-IT-MSspectrarevealednovelaspectsregardingthespeciationofcopperglutathionecomplex.KeywordsESI-MSglutathionecoppercomplexpHIntroductionG.Drochioiuetal.Eur.J.MassSpectrom.1971752013Received7February2013nAccepted18February2013nPublication26February2013 72MSApproachtoHighpHandCopper-InducedOxidationMerckDarmstadtGermany.EthanolaminewaspurchasedfromMerckSchuchardt-HohenbrunGermany.Onmixing100Lof2.50.4MpH11.8solutionofethanolaminewith100Lof1mMglutathione20Lor100Lof10mMcopperchlorideandwaterupto1mLthepHvalueoftheresultingsolutionwas11.2.ForMSmeasure-mentstheobtainedsolutionwas110dilutedwithwaterwhenpHchangedto10.950.05.Separatelythesolutionofethanolaminewasreplacedwith100LofammoniumbicarbonatebufferpH6.6.HerewereportonlytheoxidationofGSHatpHinexcessof10andcopperbindingtoglutathioneatpH6.6and12and110glutathionecoppermolarratios.ResultsanddiscussionGlutathionewasmostlyfoundinitsreducedandprotonatedforminthegas-phaseasbeingproducedinslightlyacidicsolutionsFigure1.TwomainpeakshavebeenobservedintheESI-IT-MSspectrumofglutathioneatpH6.6whichwereassignedtoprotonatedglutathioneGSHHanditsdimer2GSHHrespectively.Howeveranothersmallsignalatmz613.1wasfoundandassignedtooxidizedglutathioneGSSGHdemonstratingthehightendencyofGSHtoundergooxidationInsertB.Thepeakheightatmz309.0wasonly13.4ofthatatmz308.0whichwasassignedtoGSHH.Howeverthepeakheightatmz616.1was25.4ofthatatmz615.1whereasthepeakheightatmz613.1wasabout31.0.WeassumedthattheMSspectrumcangiveinformationonbothGSHdimerizationanditsreactiontooxida-tionpassingthroughtheradicalformGSH2GSHGSGSHGSSGmz308mz615mz616mz613.Theadditionofethanolamineinsteadofammoniumbicar-bonateenhancedthepHrangeofmassspectrometryanalysisFigure1b.EthanolamineshowednointerferencewiththemassspectrometryanalysismadewithanESIiontrapsystem.OnmeasuringpeptidesbymassspectrometrynosignalsatmzcorrespondingtothefollowingionsMH612MH61or3MH61wereobserved.HoweverethanolaminepH10.9greatlyincreasedtheproportionofoxidizedmolecules.NotracesofreducedGSHwereobservedathighpHinthepres-enceofethanolamine.Copperionsalsoinducedglutathioneoxidationasthepeaksatmz613.1GSSGHandmz307.0GSSG2H2demonstratedinFigure2a.OnstudyingtheisotopicdistributionofGSHinsertsAandBaswellastheshapeofthepeaksofcopperGSHcomplexesInsertCandcomparingitwiththetheoreticaldistributioninsertDonecancalculatetheFigure1.ESIiontrapMSspectraofglutathioneatvariouspHvaluesaglutathione10pmolL1pH4.0bglutathione10pmolL1pH10.9. G.Drochioiuetal.Eur.J.MassSpectrom.197175201373proportionooxidizedorreducedglutathioneandthepropor-tionoCuIandCuIIboundtoGSH.Thepeakatmz674.0wasclearlyassignedtothe63CuIIGSSGHionwhereasthe65CuIIGSSGHionwasoundatmz676.0.Howeverweoundasmallproportiono63CuIGSSGspeciesatmz675.0thepeakheightproportionsmz67410067530.767660.767716.2and6786.3.ThereoreonceoxidizedGSHmayreactespeciallywithCu2ionsandlesswithCuions.OnincreasingthecopperGSHratioupto101thepeakheightatmz675.0decreasedto26.8othatatmz674.0Figure2bInsertC.Thepeakatmz677.0wasveryweaksuggestingthelackoCuIGSH2complexcontrarytotheindingsintheliterature.6ThehighconcentrationoCu2inthesystemcouldexplainthelowratiobetweenCuIGSH2andCuIIGSSGHions.Insteadthepeakheightatmz735.9was37.9othatatmz734.9InsertD.Hencethesignalatmz736.9cannotbeassignedtoGSSG2Cu3HbuttoamixtureoionssuchasGSSG263CuII3HGSSG63CuII63CuI2HGSSG265CuII3HGSSG263CuIHetc.DiscussionOurresultssupportthefndingsinliteratureontheormationoacopperglutathionecomplexwhichisstableinthepresenceosomecopper-chelators.8GSHisanintracellularreducingagentorCu2ionsandacomplexationagentorCuions.9TracesoCu2ionsreadilycatalyzeoxidationoGSHandduringthesereactionsthereisaormationothiylandhydroxylradicals.1Hand13CNMRstudiesoCu2bindingtooxidizedglutathioneGSSGinaqueoussolutionoverthepHrange411showedthattheyormacomplexwitha11CuGSSGstoi-chiometry.10NeverthelesspH-dependentprecipitateswithaCu2GSSGstoichiometryarealsopossible.HoweverweoundCu2GSSGinthegas-phaseandconsequentlyCuII2GSSGmaybepresentinsolutionsatthephysiologicalpH.ItwasshownthatonlyGSHisabletoreducetheCuIIboundtoGSSG.5WedidnotfndsuchareactionprobablyduetohighconcentrationsoCu2whichmaskit.HoweversomeauthorshaveconirmedthatCu2ionsareabletoreactevenwithsimplerpeptidessuchastetraglycinetoormbothcopperIandcopperIIcomplexesinsolution.1112IthepeptidescontaincysteineresiduesthenasimilarredoxreactiontothatoundinGSHmayoccur.1316InSaccharomycescerevisiaeCd2ionsalsoreactwithGSHormabisglutathionatocadmiumCdGS2complexwhichcanbetransportedromthecytosolintothevacuole.17GSHisastrongreducingagentandisabletoreducemanyCuIIcomplexeswhichentercells.9WealsoassumedthathighconcentrationsoCu2aredetoxifedbyoxidizingGSHwhichFigure2.ESIiontrapMSspectraofglutathione-coppercomplexesatpH6.6inthepresenceofammoniumbicarbonateaglutathione10pmolL1glutathionecopperion12bglutathione10pmolL1glutathionecopperion110. 74MSApproachtoHighpHandCopper-InducedOxidationisstoredinvacuolesorremovedromthelivingbodiesasCuIIGSSGcomplexes.IncontrastmercurydoesnotoxidizeGSHmz308.0peakinthepresenceoHg2butinducestheormationoGSHHgHmz508.0GSHgGSHmz813.1orMHgClHmz544.0toreduceHgtoxicity.1819MSstudiesrevealedgreatbindingdierencesamongheavymetalionswhichalsodependedoncysteinepresenceorabsence.2022InbrieglutathioneisreadilyoxidizedevenunderslightlyacidicconditionsasMSspectrashowed.UnderanalkalineenvironmentorinthepresenceoCu2ionsGSHiscompletelyoxidizedtoGSSG.TheoxidizedglutathionereactswiththeexcessoCu2ionsandlesswithCuions.MSspectrarevealedthepresenceobothCuIandCuIIcomplexesoGSHinwhichmetalionsaremoreprobablycomplexedwithaminoandcarboxylgroupsthanboundtoSHions.Suchhypothesisissupportedbysomefndingsintheliteraturewhichshowthatcopperionsbindtoaminogroupsandtheunstructuredregioncontainingglycineresidues.2324FurtherresearchisneededtorevealcopperdetoxifcationmechanismswhichinvolveGSH.AcknowledgmentFundingromRomanianGovernmentUEFISCDIIDEI3132011.References1.G.Y.KreitmanV.F.LaurieandR.J.EliasInvestigationoethylradicalquenchingbyphenolicsandthiolsinmodelwineJ.Agric.FoodChem.616852013.doi10.1021j303880g2.Y.HatoriS.ClasenN.M.HasanA.N.BarryandS.LutsenkoFunctionalpartnershipothecopperexportmachineryandglutathionebalanceinhumancellsJ.Biol.Chem.287266782012.doi10.1074jbc.M112.3811783.J.R.WintherandU.JakobRedoxcontrolablackholeoroxidizedglutathioneNat.Chem.Biol.9692013.doi10.1038nchembio.11614.T.PivettaF.IsaiaG.VeraniC.CannasL.SerraC.CastellanoF.DemartinF.PillaM.MancaandA.PaniMixed-110-phenanthrolineCuIIcomplexesSynthesiscytotoxicactivityversushematologicalandsolidtumorcellsandcomplexormationequilibriawithglutathioneJ.Inorg.Biochem.114282012.doi10.1016j.jinorg-bio.2012.04.0175.M.E.AliagaC.Lpez-AlarcnL.Garcia-RioM.Martn-PastoandH.SpeiskyRedox-changesassociatedwiththeglutathione-dependentabilityotheCuII-GSSGcomplextogeneratesuperoxideBioorg.Med.Chem.2028692012.doi10.1016j.bmc.2012.03.0276.H.SpeiskyM.GmezF.Burgos-BravoC.Lpez-AlarcnC.JullianC.Olea-AzarandM.E.AliagaGenerationosuperoxideradicalsbycopperglutathionecomplexesredox-consequencesassociatedwiththeirinteractionwithreducedglutathioneBiorg.Med.Chem.1718032009.doi10.1016j.bmc.2009.01.0697.A.R.WhiteandR.CappaiNeurotoxicityromgluta-thionedepletionisdependentonextracellulartracecopperJ.Neurosci.Res.718892003.doi10.1002jnr.105378.I.JimnezandH.SpeiskyEectsocopperionsonthereeradical-scavengingpropertiesoreducedgluthathioneimplicationsoacomplexormationJ.TraceElem.Med.Biol.141612000.doi10.1074jbc.M110.1544689.A.CorazzaI.HarveyandP.J.Sadler1H13C-NMRandX-rayabsorptionstudiesocopperIglutathionecomplexesEur.J.Biochem.2366971996.doi10.1111j.1432-1033.1996.0697d.x10.W.S.PostalE.J.VogelC.M.YoungandF.T.GreenawayThebindingocopperIIandzincIItooxidizedgluta-thioneJ.Inorg.Biochem.25251985.doi10.10160162-01348583004-X11.G.SchlosserR.SteanescuM.PrzybylskiM.MurariuF.HudeczandG.DrochioiuCopper-inducedoligomerizationopeptidesamodelstudyEur.J.MassSpectrom.133312007.doi10.1255ejms.88912.M.MurariuE.S.DraganandG.DrochioiuIRMSandCDinvestigationsonseveralconormationally-dierentpeptidesInt.J.Pept.Res.Therap.153032009.doi10.1007s10989-009-9192-113.M.MurariuE.S.DraganandG.DrochioiuSynthesisandmassspectrometriccharacterizationoametal-afnitydecapeptidecopper-inducedconormationalalterationsBiomacromolecules838362007.doi10.1021bm700793g14.M.MurariuK.PopaE.S.DraganandG.DrochioiuSyntheticcysteine-peptideanditsrelationwithheavyandradioactivemetalsRev.Roum.Chim.547412009.15.G.DrochioiuM.MurariuB.A.PetreM.ManeaandM.PrzybylskiSynthesisandcharacterizationoanonapeptidewithspecifcCu-bindingpropertiesRev.Chim.Bucharest583112007.16.G.DrochioiuM.ManeaM.DragusanuM.MurariuE.S.DraganB.A.PetreG.MezoandM.PrzybylskiInteractionob-amyloid140peptidewithpairsometalionsanelectrosprayiontrapmassspectrometricmodelstudyBiophys.Chem.14492009.doi10.1016j.bpc.2009.05.00817.A.A.Mielniczki-PereiraA.Z.SchuchD.BonattoC.F.CavalcanteD.S.VaitsmanC.J.RigerE.C.AraujoEleutherioandJ.A.P.HenriquesTheroleotheyeastATP-bindingcassetteYc1pinglutathioneandcadmiumionhomeostasisduringrespiratorymetabolismToxicol.Lett.180212008.doi10.1016j.toxlet.2008.05.01018.R.GradinaruA.IonasA.PuiG.ZbanciocandG.DrochioiuInteractionoinorganicmercurywith G.Drochioiuetal.Eur.J.MassSpectrom.197175201375CoA-SHandacyl-CoAsBiometals2411152011.doi10.1007s10534-011-9472-z19.K.PopaM.MurariuG.SchlosserR.MolnarA.CecalandG.DrochioiuEffectofradioactiveandnon-radioactivemercuryonwheatgerminationandtheanti-toxicroleofglutathioneIsot.Environ.HealthS.21052007.doi10.10801025601070136211220.M.MurariuE.S.DraganandG.DrochioiuModelpeptide-basedsystemusedfortheinvestigationofmetalionsbindingtohistidine-containingpolypeptidesBiopolymers934972010.doi10.1002bip.2138521.M.MurariuE.S.DraganandG.DrochioiuElectrosprayionizationmassspectrometricapproachofconformationally-inducedmetalbindingtooligopeptidesEur.J.MassSpectrom.165112010.doi10.1255ejms.109222.G.DrochioiuAnelectrosprayionizationmassspectrometricstudyofironbindingtoamyloid-bpeptide.Eur.J.MassSpectrom.156512009.doi10.1255ejms.100423.G.DrochioiuM.MurariuI.MangalagiuandI.DrutaHighlyselectiveassayofproteinsindilutesolutionsTalanta564252002.doi10.1016S0039-91400100562-824.W.L.PangA.KaurA.V.RatushnyA.CvetkovicS.KumarM.PanA.P.ArkinJ.D.AitchisonM.W.AdamsandN.S.BaligaMetallochaperonesregulateintra-cellularcopperlevelsPLoSComput.Biol.9e10028802013.doi10.1371journal.pcbi.1002880 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