| ABSTRACT |
| ForMALDI-TOF-MSanalysisofproteinsandpeptides,samplesarecocrystallizedwithanexcessoforganicmatrixthatabsorbsataspecificwavelength(usually,UV337nm).Typically,sinapinicacid(SA)isthematrixofchoiceforlargeproteins,whereas{alpha}-cyano-4-hydroxy-cinnamicacid(HCCA)isthepreferredmatrixforpeptidemapping.Followingashortlaserpulse,analytesareprotonatedanddesorbedintothegasphase,andtheirm/zvaluesaredeterminedinaTOFmassanalyzer.Massaccuracydeterminationsvaryfrom±0.01%to0.1%dependingonthesamplepreparationtechniqueandthemethodusedformasscalibration. |
| MATERIALS |
| Reagents |
- Calibrationstandards(1-10pmol/µlin60%acetonitrilecontaining0.1%[v/v]TFA)
- ToobtainthebestinterpretationofMALDI-TOF-MSdata,standardsofknownmolecularmassclosetothemolecularmassoftheunknownsamplearerequiredtoensurealinearcalibrationcurve.Thesemustbepreparedandchosencarefully.Typically,angiotensinIorangiotensinII,whoseaccuratemolecularmassesareknown,canbeusedforpeptidemappingstudies.Table2andTable3containarangeofpeptidesandproteinsthatcanbeusedroutinelyasstandardsinMALDI-MSanalysis.Allofthesestandardsarecommerciallyavailable,andtherefore,stocksolutionscanbeaccuratelyprepared.Stocksolutionsofpeptidecalibrantsarepreparedataconcentrationof100pmol/µlaliquots(frozenstocksaremadeupin60%acetonitrilecontaining0.1%TFA,andcanbekeptforseveralyears).Forworkingsolutions,thesealiquotsaredilutedto1-10pmol/µlwithaqueous0.1%(v/v)TFA.Workingsolutionsofcalibrantcanbeusedfor1weekwhenkeptat4°C.Theconcentrationofthecalibrationstandardsmustbesimilartothatoftheunknownsample(range1-10pmol/µl)toensureaccurateresults.
- 60%acetonitrilecontaining0.1%[v/v]TFA
- Lyophilizedsamples(dissolvedin60%acetonitrilecontaining0.1%[v/v]TFAataconcentrationof1-10pmol/µl)orRP-HPLC-purifiedfractions,whichcanbeuseddirectly
- Samplescontainingexcipientssuchasbuffers,salts,detergents,anddenaturantsmustbedesaltedpriortoanalysis.Evenminorquantitiesofsodium(m/z23)andpotassium(m/z39)ions,reADIlygeneratedvialaserionization,cancausesignificantionsuppression.Thedetergentn-octylglucoside(0.1%)canbeaddedatdifferentstagesofsamplepreparation(e.g.,digestionorsolubilizationofdigestedpeptides)topreventadsorptionofpeptidesonthesampletubewalland/orPipettetip,therebyyieldingincreasedpeptidepeaknumberandimprovedsequencecoverage.
- MALDI-MSmatrixsolution
- 20mg/mlmatrix(e.g.,HCCA,DHB,orSA)
- 60%acetonitrile
- 0.1%(v/v)TFAUndertheseconditions,thematricesaresaturatedsolutionsandmustbecentrifugedpriortouse.
Methanol |
| Equipment |
- Lint-freetissues(e.g.,Kimwipes)
- MALDImassspectrometerSeethenotetoStep6formoreinformation.
- MALDIplate
- Pipettetips,2-µl
- Oven,40°C(optional;seeStep3)
|
| METHOD |
- Pipette0.5µloftheMALDI-MSmatrixsolution(using2-µlpipettetipsforaccuracy)ontothesamplewellsofthemetalsampleplatesusedforMALDI-MSanalysis.
- Immediatelyadd0.5µlofthestandardorsampletothematrixbeforeitdries.
- Allowthesolventtoevaporateandthesamplestodryfor~5minuteseitheratroomtemperatureorina40°Coven.
- Transferthemetalsampleplatetothevacuumchamberofthemassspectrometer.
- Acquireaninitialmassspectrumatalaserpowerwellabovetheionizationthresholdto"warmup"thecalibrationstandard.
- Decreasethelaserpoweruntilagoodspectrumisobtained.Steps5and6applytoMALDI-TOFinstrumentsthatareequippedwithahigh-vacuumionizationsource(e.g.,KompactMALDIIVTM;KratosAnalytical/Shimadzu,orequivalent).ForinstrumentsequippedwithaCCDcameraforviewingthecocrystallizedsample(e.g.,o-MALDIQSTARPulsarI;AppliedBiosystems,orequivalent),selectalargecrystalforanalysis(e.g.,largecrystalsformwhenusingthematrixDHB;thisisnotsoimportantwhenusingHCCAorSA,whichusuallyyielduniformlysmallcrystals).HCCAispreferredasaMALDImatrixforpeptidemappingbecauseityieldsthehighestsensitivityandformsauniformmatrixlayeronaMALDIsampleplate,whichmakesitamenableforautomatedanalysis.Foroptimalresults,runtheunknownsamplesatapproximatelythesamelaserpowerasthecalibrationstandard.Althoughahighlaserpowermaygiveagoodsignal,resolutionmaybecompromisedduetopeakbroadening.Theuseofalowlaserpowerattheionizationthresholdmaygivehighresolution,butresultinpoorsignal-to-noiseratios.Typically,thethresholdsofionizationforHCCAandDHBare20and30µJ,respectively.Thus,HCCAisconsideredtobea"hotter"matrixthanDHB,whichgivesrisetoincreasedmetastableionformationandconcomitantPSD.Aconsequenceofthelatterisbroaderpeakformationandreducedresolution.Althoughasamplemayappeartobeuniform,itisrecommendedthatdifferentregionsofthespotbeexaminedtofind"sweetspots,"thatis,regionsofthesamplespotthatgivesuperiorsignal-to-noiseratios.
- Obtainalinearexternaltwo-pointcalibrationusinganappropriatecalibrant.WhenusingHCCA,thematrix-derivedion(e.g.,[M+H]+-OH,m/z173.2)andthesinglychargedionoftheappropriatecalibrationstandardcanalsobeusedascalibrants
- RepeatSteps5and6forunknownsamplesandcomparewiththecalibrationvaluestoobtainaccuratemasses.
- Afteruse,cleantheMALDIplatebyrinsingthoroughlywithH2OtoremoveanyvisIBLecrystalsandthenwithmethanol;wipewithlint-freetissues(e.g.,Kimwipes).
|
| REFERENCES |
| Kussmann,M.andRoepstorff,P.2000.SamplepreparationtechniquesforpeptidesandproteinsanalyzedbyMALDI-MS.MethodsMolBiol146:405-424.[Medline] |
| Anyoneusingtheproceduresinthisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliABIlityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhazardousandshouldbeusedwithcaution.Forafulllistingofcautionsregardingthesematerial,pleaseconsult:ProteinsandProteomics,ALaboratoryManual,byRichardJ.Simpson,©2003byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p.478-481. |
