药物分析英文文献15

FromExogenoustoEndogenous:TheInevitableImprintofMassSpectrometryinMetabolomicsElizabethJ.Want,AndersNordstro1m,HirotoshiMorita,andGarySiuzdak*DepartmentofMolecularBiology,TheScrippsCenterforMassSpectrometry,10550NorthTorreyPinesRoad,LaJolla,California92037ReceivedSeptember27,2006Massspectrometry(MS)isanestablishedtechnologyindrugmetaboliteanalysisandisnowexpandingintoendogenousmetaboliteresearch.Itsutilityderivesfromitswidedynamicrange,reproduciblequantitativeanalysis,andtheabilitytoanalyzebiofluidswithextrememolecularcomplexity.Theaimsofdevelopingmassspectrometryformetabolomicsrangefromunderstandingbasicbiochemistrytobiomarkerdiscoveryandthestructuralcharacterizationofphysiologicallyimportantmetabolites.Inthisreview,wewilldiscussthetechniquesinvolvedinthisexcitingareaandthecurrentandfutureapplicationsofthisfield.Keywords:massspectrometry¥liquidchromatography¥metabolomics¥biomarkercharacterization¥metabolitedatabaseIntroductionTheapplicationofmodernmassspectrometrytechnologytoendogenousmetaboliteresearchderivesfromitssuccessindrugmetabolitestudies,bothquantitativeandstructural.1-11Interestalsooriginatesfromtheabilitytoperformmorecomprehensivemetaboliteanalyseswithnewliquidchroma-tography/massspectrometry(LC/MS)technologies,suchasnanoESI-LC/MS,andthedesiretounravelbasicbiochemicaleventsofcellsandtissues,ortoidentifydiseaseorpharma-ceuticalbiomarkers.OneofthefirstmetaboliteprofilingexperimentswasbyPaulingandcolleaguesin1971,whoanalyzedthemetabolitecontentofhumanurinevaporandbreathofsubjectsonadefineddietusinggaschromatography(GC).12Approximately250substancesweredetectedinabreathsampleand280inaurinevaporsample.Thisgroupthenwentontoprofileaminoacidsinurine,employingnonparametricstatisticalanalysisfordetectingprofiledifferencesrelatedtogenderandothervariables.13Thiswasthebeginningofwhatwenowcallmetabolomics,theaimofwhichistoprovideacomprehensiveprofileofallthemetabolitespresentinabiologicalsample.Fromthe1970s,gaschromatographymassspectrometry(GC/MS)becamepopularformetaboliteprofilingandisstillusedforthedetectionofmanymetabolicdisorders.14Advan-tagesofGC/MSincludehighresolutionandreproducibility,aswellastheavailabilityofEIspectrallibrariesforstructuralidentification.15Inaddition,sincethe1990s,16nuclearmagneticresonance(NMR)hasalsobeenappliedtoareassuchasplantmetabolism,DuchenneMuscularDystrophy,neurologicaldis-orders,andhepatotoxicityandnephrotoxicityinrodents,17-24withadvantagesinbothspeedandaccuracy.However,becauseofthelimitationsofNMRintermsofsensitivity,LC/MShasemergedasapowerfulalternativetechnologyformetabolom-ics.Inthisreview,theroleofmassspectrometryinmetabo-lomicswillbediscussed,encompassingdataacquisition,dataanalysis,metabolitecharacterization,andmanyexcitingap-plications.1.DataAcquisitionDuetothecomplexnatureofbiologicalsamples,separationisoftenperformedbeforemassspectrometricanalysistoachievethedetectionofasmanymetabolitesaspossible.Traditionally,GCwasemployed,asitiswell-knownforhighresolutionandreproducibility.However,disadvantagesofGCincludeconvolutedsamplepreparation(suchasderivatization),lengthyanalysistime,andthelimitationonthesizeandtypeofmoleculethatcanbeanalyzed(nonvolatile,polarmacro-moleculesareunsuitable).However,GC-MSisstillwidelyusedinplantmetabolomicsdueinparttothenatureofthemetabolitesbeinginvestigated.15,25-28Liquidchromatographyelectrosprayionizationmassspec-trometry(LC/ESI-MS)(Figure1)isnowacommonmetabolo-micstool.SeparationofthethousandsofmoleculespresentinbiofluidsusingLCcanreduceionsuppression29-31bydecreasingthenumberofcompetinganalytesenteringthemassspectrometerionsourceatanyonetime.Thisresultsinaselectiveapproachthatallowsforbothquantitationandstructuralinformation,wheresensitivitiesinthepg/mLrangecanbeachievedreadily.32LC/MStechniqueshavereplacedsomeofthetraditionalspecializedclinicallaboratorymeth-ods33,34thatusedimmunological,fluorometric,andbiologicaltechniques.35AnimportantfactorinLCmetaboliteseparationisthechoiceofcolumn.Manybiofluids,particularlyurine,containavastarrayofhighlypolarmoleculesthatarenotretainedwellonthemoretraditionalreversephase(RP)LCcolumns.Normalphasetechniques,whichresultintheelutionoflesspolarmoleculesfirstandthustheretentionofmorepolarmolecules,10.1021/pr060505+CCC:$37.002007AmericanChemicalSocietyJournalofProteomeResearch2007,6,459-468459PublishedonWeb11/18/2006requireadifferentsolventsystemtothatusedbyRPchroma-tography,typicallycontainingnoaqueous.Anewerapproachishydrophilicinteractionchromatography(HILIC),whichcanoffercomplementaryinformationtothatobtainedusingRPchromatography.36Here,waterandacetonitrilecanstillbeused,althoughstartingfromahighorganiccontentandendingathighaqueous.HILICapproachescombinedwithESI-MStechniqueshavealreadybeenappliedtotheanalysisofdichloroaceticacidinratbloodandtissues,37plantmetabolitessuchasoligosaccharides,glycosides,andsugarnucleotides,38andwithAPCImassspectrometryforthedeterminationof5-fluorouracilinplasmaandtissues.39TheabilityofLCtoseparatecomplexmixturespriortomassanalysiscomesatacostofspeed.Analternativetotradi