DenovosequencingofpeptidesbyMS/MSJoergSeidler,NicoZinn,MartinE.Boehman - PDF document

DenovosequencingofpeptidesbyMS/MSJoergSeidler,NicoZinn,MartinE.BoehmandWolfD.LehmannMolecularStructureAnalysis,GermanCancerResearchCenter,Heidelberg,GermanyReceived:June30,2009Revised:September18,2009Accepted:September23,2009ThecurrentstatusofdenovosequencingofpeptidesbyMS/MSisreviewedwithfocusoncollisioncellMS/MSspectra.TherelationbetweenpeptidestructureandobservedfragmentionseriesisdiscussedandtheexhaustiveextractionofsequenceinformationfromCIDspectraofprotonatedpeptideionsisdescribed.ThepartialredundancyoftheextractedsequenceinformationandahighmassaccuracyarerecognizedaskeyparametersfordenovosequencingbyMS.Inaddition,thebeneÞtsofspecialtechniquesenhancingthegenerationoflonguninterruptedfragmentionseriesfordenovosequencingarehighlighted.AmongtheseareterminalOlabeling,MSofsodiatedpeptideions,N-terminalderivatization,theuseofspecialproteases,andtime-delayedfragmentation.TheemergingelectrontransferdissociationtechniqueandtherecentprogressofMALDItechniquesforintactproteinsequencingarecovered.Finally,theintegrationofbioinformatictoolsintopeptidedenovosequencingisdemonstrated.bionseries/CID/Manualsequencing/Sequenceinformation/yionseries1IntroductionTheintroductionofchemicalproteinsequencingbyEdmandegradation[1]inthe1950swasamilestoneinthedevel-opmentofproteinresearch.ForidentiÞcationofEdmandegradationproducts,mainlyLCwasused.Intermittently,MSwasintroducedasalternativemethodtoLCwithopticaldetection.Thiswasachievedincombinationwiththeearlysoftionizationtechniqueschemicalionization[2],Þelddesorption[3],andfastatombombardment[4].Later,thegradualreÞnementofMS/MStechniquescreatedthebasisforpeptidesequencingbyMS(forareview,see[5]),whichÞnallygavefastaccesstomultipleinternalproteinsequen-cesbytheanalysisofproteolyticpeptides[6Ð8].Inaparalleldevelopmenttermedladdersequencing,proteinsequenceswerereadbysinglestageMALDI-MSfromsequenceladdersgeneratedbyexopeptidases[9Ð12].Withinthelasttwodecades,proteinsequencedeterminationbyMS/MSbecamemoreandmorepowerful,adevelopmentdrivenmainlybytheimprovementsofLCtechniquesincombi-nationwithESI[13].TheadvantagesofMS/MStechniqueswithrespecttospeed,sensitivity,andapplicabilitytocomplexpeptidemixturesgraduallyledtothereplacementofEdmantechniquesbyLC-MS/MS.Asaresult,MS-basedproteomicshasemergedasthemethodofchoicefortheidentiÞcationofproteins[14,15]interpretationofMSdatausingsearchenginessuchasMASCOT[16],SEQUEST[17],X!TANDEM[18],orOMSSA[19].MSdataaretypicallyeitherMALDImassÞngerprint Correspondence:ProfessorWolfD.Lehmann,MolecularStruc-tureAnalysis(W160),GermanCancerResearchCenter,ImNeuenheimerFeld280,69120Heidelberg,Germanywolf.lehmann@dkfz.de49-6221-42-45-542010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.proteomics-journal.com,634–649DOI10.1002/pmic.200900459 presentinthereferencedatabase.Inspiteofthecontinu-ouslygrowingsequencedatabases,denovosequencingofsequencingwithoutassistanceofalinearsequencedatabase,isstillessentialinseveralanalyticalsituations.Forexample,analysesofproteinsequencevariantsortheirspliceisoformsrequiredenovoaswellasproteinanalysisfromorganismswithunse-quencedgenomes.Inaddition,denovosequencingisessentialforanalysisofpeptidescontainingnon-proteinicormodiÞedaminoacids,astypicallypresent,inbioactivepeptidesofbacteriaorfungi[24].TheperformanceofboththeMS/MSandoftheLCpartinßuencestheutilityofanLC-MSsystemfordenovosequencing.ThisisbecausethesigniÞcanceofpeptideMS/MSdataisconnectedwiththepurityofthepeptideionsselectedforfragmentation.Inthefollowing,thestate-of-the-artandcurrentadvancementsofpeptidedenovosequencingbyMSarereviewedanddiscussed.ImportantpointsaretheredundancyofsequenceinformationpresentinCIDspectra,theuseofnewpeptideactivationtechniques,MSanalyses,theinßuenceofmassaccuracy,labelingtechniques,thefragmentationofintactproteins,andbioinformatictoolsforareference-freeinter-pretationofMS/MSspectra.2NomenclatureofpeptidefragmentionsTheintroductionofsoftionizationtechniquesenabledtheefÞcientgenerationofintactpeptideionswhichcanbeselectedasprecursorsforsubsequentactivationanddetec-tionoftheirfragmentions.Forpeptidesequencing,thepositivemodeisgenerallyusedduetoitshighersensitivityandsinceMS/MSspectraofprotonatedpeptidescontainawealthofsequence-speciÞcfragmentions.Incontrast,negativeionMS/MSspectracontainlesssequenceinfor-mationandareusuallymoredifÞculttointerpret.Frag-mentationofprotonatedpeptideionsfollowingCIDoccurspredominantlyatthepeptidebackbonebyproton-inducedfragmentationreactionsasexplainedbythemobileprotonmodel[25].ThewidelyacceptednomenclaturefortheannotationofpeptidesequenceionsisoutlinedinFig.1,asoriginallyproposedbyRoepstorffandFohlman[26]andlatermodiÞedbyBiemann[27].SequenceionsthatundergoneutrallossaredistinguishedfurtherasforexamplethelossofHOisindicatedbyorthelossofNH3CIDThemajorityofpeptidedenovosequencinghasbeenperformedusingCID(orcollision-activateddissociation).InlowenergyCID,activationofmolecularionsisachievedbycollisionswithinertgasmolecules(He,N,Ar)presentinaseparatecollisioncellorasbathgasinaniontrap.InCID,thetranslationalenergyoftheionsispartiallyconvertedintointernalvibrationalenergywhichtheninducespeptidefragmentation.Accelerationoftheprecursorionsinthecollisionzonebyapotentialdifference(collisionoffset)intensiÞesthefragmentationandleadstomoresecond(orhigherorder)fragments,duetorepeatedcollisionsduringthetravelthroughthecollisioncell.Moderatecollisionoffsetvaluesaremostfavorableforsequencing,intermsofabsolutefragmentionintensityandbackgroundlevel.ForunmodiÞedpeptides,activationofprotonatedmoleculesleadsprimarilytobackbonefragmentations,resultinginstructure-speciÞcfragments.Themobileprotonmodel[25]cansemi-quantitativelyrationalizethedistributionoftheobservedfragments[28,29].Aparticularfeatureofback-bonecleavagesofmultiplyprotonatedmolecularionsisthattheymayresultincomplementaryb/yfragmentions.CleavagesattheN-terminalsiteofPorattheC-terminalsiteofDaretheprototypeformsoffragmentationresultingincomplementaryfragmentions.However,incollisioncellMS/MSspectraofpeptidestheoccurrenceofcomplemen-taryionsisrathertheexceptionthantherule.ThisimpliesthatthemajorityofbackbonecleavagesareonlyrepresentedbyeitherboryionsandthattheindividualC-orN-terminalfragmentionseriesmostlyexhibitonlyaminoroverlap.ThissituationisdemonstratedinFig.2foracollectionofquadrupoleTOF(Q-TOF)CIDspectrasummarizingtheirsearchengine-annotatedfragmentionseries.ThedatainFig.2implythatthelocationofthebasicresiduesR,K,andHdeterminesthebalancebetweenthelengthofthebandyionseries.TheCIDspectraoftypicaltrypticpeptideswithabasicresidueattheC-terminusarecharacterizedbyextendedyionseriesaccompaniedbyshortbionseries(btobwithoutbions(Fig.2A).PeptideswithabasicresidueattheN-terminusmaybegenerated,bydigestionwithLysN.TheirCIDspectraarecharacterizedbylongbionseriesinconnectionwithshortyionseries(Fig.2B).PeptideswithbasicresiduesatbothterminiexhibitCIDspectrawithbandyionseriesofcomparablelengthoftenwithmoderateoverlap(Fig.2C),andpeptideswithtryptic 2N O1R3R2R4 O yx abc Figure1.Nomenclatureofsequence-specicpeptidefragments;a-,b-,andc-typeionscontaintheN-terminus;x-,y-,andz-ionscontaintheC-terminus;hydrogenrearrangementsareomittedinthissimpliedannotation(accordingto[27]).R1,R2,R3repre-sentthesidechainsoftheaminoacidresidues.,634–6492010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.proteomics-journal.com miscleavagesitesshowasimilarcharacteristic.Ingeneral,theiroccurrenceiscausedbyeitheraKK,RR,KR,RK,KP,orRPmotiforbythepresenceofanacidicresidueinoneofthesequencepositions3relativetoKorR[32,33].Forthesepeptideswithinternalbasicresidues,amorepronouncedmutualoverlapofthefragmentionseriesorsequenceionsuppressionaroundinternalbasicresiduesmayoccur(Fig.2D).Inpractice,backbonecleavageinthevicinityofRandKresiduesisnotveryeffectiveduetosequesteringoftheprotonsatthebasicsite.Thus,thecorrespondingionsareoflowrelativeabundance,sothattheymayescapedetection,asobservedforthepeptideinternalKstartingwithQES(Fig.2D).ThepresenceoftwoormoreRresiduesindirectorclosevicinitynormallycausespronouncedgapsinfragmentionseriesofanytype,duetothehighbasicityofR.ESIpeptideionswithchargestate2and3arethepreferredprecursorionsfordenovosequencing.Inpractice,theMS/MSspectraofallaccessiblechargestatesshouldberecorded,sincethecorrespondingspectraoftenexhibitboryionseriesofdifferentlength,sothatdifferentregionsofthepeptidemaybecomeaccessibleinthisway.Ingeneral,fragmentionspectraofhigherchargestatescontainmoresequenceions;however,MS/MSspectraofdoublychargedionsareoftenmoreeasilyinter-pretedthanthoseoftriplyorquadruplychargedprecursorions.Themanualannotationofthebasicionserieshasbeenexplicitlysummarizedinastep-by-steptutorial[34].Besidesbandyions,additionaltypesoffragmentionsoccurinCIDspectraofpeptideswhichalsocontainsequenceinformation.Thesecompriseneutrallossreac-tionsfromthepeptidetermini[35,36]andinternalfrag-mentions[37].ThisisexempliÞedforthedenovosequencingofthepeptideSNTDANQ[L/I]WT[L/I]KshowninFig.3.ThispeptidewasobtainedbytrypticdigestionofatypeIIribosome-inactivatingproteinfromtheplant.Itssequencecouldbedeterminedonthebasisofthealmostcompleteyionseriesfromytoy(Fig.3A).Inaddition,acontinuousbionseriesisobservedfromtob).Inspectionofthelowmasssideofthethe12H]21signalrevealedtheoccurrenceofneutrallossfragmentations,whichcanbeeasilyrecognizedbytheirchargestate(Fig.3B).TheseionsshowtheN-terminallossofSfollowedbythelossofN,whichresolvestheambiguityinthestructureofthebionat202betweenSNandNS.TheoccurrenceoftheN-terminalsequenceSNisalsosupportedbythebionfragmentproÞle.ThisproÞleshowsthelossofNH),whichistypicalforbwhereasbNSionsshowthepreferentiallossofH(referencedatanotshown).Theassignmentofsequenceandcompositionalisomersofbtheirfragmenta-tionproÞleshasbeendemonstratedrecently[38,39].ThelowmassregionoftheMS/MSspectrum(Fig.3C)alsoprovidescompositionalinformationintheformofimmo-niumions.Table1summarizesthesequenceandcompositionalinformation,whichcanbeextractedfromtheMS/MSspec-truminFig.3.Itcanberecognizedthatredundantinfor-mationisobtainedfortheN-terminusbasedonthebfragmentationproÞleandontheneutrallossesfromthemolecularion.ThecentralsequenceTDANQisbasedbothontheoccurrenceofbandyions.Finally,theoccurrenceoftheC-terminalKresiduesissupportedbothbythefragmentionmassandbythespeciÞcityoftrypsin.TheresultisroundedbythedetectionoftheimmoniumionsforQ,W,and[L/I]. T V D ME S T E V F T F Q S E E Q Q Q T E D E L Q D V P Q L E I V P N S A E E S L Y S S S P G G A Y V T Q E S L S P E E E D Q Q A F L G G D V S P T Q I D V S Q F G S F G W S E S E Q S E E F G G G I A T oxME S E D G V E G D L G E T Q S L G G S G S V V P G S P S L D I E G A S L E L S D D D T E S S P S W E P F D W Y P A E E S E E S E D D M G F G L F D S T Q G V T L T D L Q E A E S L P N S L D Y A Q A S E G S E Q E S V E F L A F P S F S S D L T L G T G S F I E D V G S D E E D D S G E S D D P E I E D V G S D E E E E E Q L S S G V S E I T V D ME S T E V F T T V D ME S T E V F T F Q S E E Q Q Q T E D E L Q D F Q S E E Q Q Q T E D E L Q D V P Q L E I V P N S A E E V P Q L E I V P N S A E E S L Y S S S P G G A Y V T S L Y S S S P G G A Y V T Q E S L S P E E E D Q Q A F L G G D V S P T Q I D V S Q F G S F L G G D V S P T Q I D V S Q F G S F G W S E S E Q S E E F G G G I A T oxME G W S E S E Q S E E F G G G I A T oxME S E D G V E G D L G E T Q S S E D G V E G D L G E T Q S L G G S G S V V P G S P S L D L G G S G S V V P G S P S L D I E G A S L E L S D D D T E S I E G A S L E L S D D D T E S S P S W E P F D W Y P A E E S E E S E D D M G F G L F D E E S E E S E D D M G F G L F D S T Q G V T L T D L Q E A E S T Q G V T L T D L Q E A E S L P N S L D Y A Q A S E S L P N S L D Y A Q A S E G S E Q E S V E F L A G S E Q E S V E F L A F P S F S S D L F P S F S S D L T L G T G S F I E D V G S D E E D D S G I E D V G S D E E D D S G E S D D P E I E D V G S D E E E E E E S D D P E I E D V G S D E E E E E Q L S S G V S E I Q L S S G V S E I Figure2.Distributionofb()andy(seriesfragmentionsinQ-TOFCIDspectraasobservedinasetofpeptideswithdifferentdistributionsofbasicaminoacids(datafromfromdatafrom[31]);(A)peptideswithabasicresidueattheC-terminus;(B)peptideswithabasicresidueattheN-terminus;(C)peptideswithbasicresiduesatbothtermini;(D)peptideswithterminalandinternalbasicresidues.(ExplanationoftheannotationforQLSSGVSEIR:thearrowsindicatethepresenceofbandoftheyseriesfromyJ.Seidleretal,634–6492010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.proteomics-journal.com Complementaryb/yionpairsdonotonlygivesequenceinformationbutalsoprovideadditionalmolecularweightinformation,sincethesumoftheirmassvaluesisequivalenttotheprecursorionmass.Thisishelpfulincaseamixtureofprecursorionsoraprecursorionofverylowabundanceisisolated.InadditiontothesequenceinformationsummarizedinTable1,peptideMS/MSspectrasometimesshowinternalbtypefragmentions[37].Finally,reporterfragmentationsforcovalentmodiÞca-tionsarehighlyusefulforrecognitionofmodiÞedpeptides[40].Overall,theoccurrenceofredundantsequenceinfor-mationcontainedinseveraltypesoffragmentationsofmultiplychargedionsisabeneÞcialfeatureofESIincombinationwithcollisioncellCID,whichstronglyfavorsitsusefordenovosequencingofpeptides.CollisioncellMS/MSspectraarehighlydependentonthecollisionoffsetused.Theextentoffragmentationmaybevariedfromonlypartialtocompletedecompositionofthemole-cularionbyincreasingtheoffsetvalue.Simultaneously,fragmentionsundergosecondary(andhigherorder)frag-mentations.Nevertheless,duetothelargebodyofexperi-mentalMS/MSdataavailable,optimaloffsetvaluescanbeselectedempirically.4ComparisonofcollisioncellandiontrapCIDPeptidefragmentionspectrageneratedinacollisioncellorinaniontraparebothgeneratedbyCID.Nevertheless,theyexhibitdifferencescausedbythewayofactivation,massanalysis,andtimescale.Inprinciple,iontrapMS/MSspectraareveryreproducibleanddominatedbyÞrstgenerationfragments,sincefragmentsarenormallynotfurtheractivatedduetotheirdifferentvalue.Thus,typicallymorecomplementaryb/yionpairsareobservedcomparedtoQ-TOFMS/MSspectra.Thisleadstotheoccurrenceofpronouncedoverlapsbetweenbandyionseries,evenfornormaltrypticpeptideswithasinglebasicresidueattheC-terminus(Fig.4).Thischaracteristiccreatesredundantsequenceinformation,whichputstheread-outofthesequenceonareliablebasis. Figure3.DenovobyQ-TOFCIDofthetrypticpeptideSNTDANQ[L/I]WT[L/I]KoriginatingfromatypeIIribosome-inactivatingproteinisolatedfromXimeniaamer-;(A)completeMS/MSspectrumcharacterizedbyyandbionseries;(B)expandedcentralregionshowingC-terminalneutrallosses;(C)expandedlowmassregionshowingthey,andthebionanditsfragments,aswellasimmoniumions.,634–6492010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.proteomics-journal.com denovosequencing,iontrapMS/MSspectrahaveacertainlimitationofaÔÔlowmasscut-off,ÕÕwhichisdirectlyproportionaltothevalueoftheprecursorion.Theaveragesizeoftrypticpeptidesexcludesfragmentiondetectionbelowabout200Ð300,limitingtheinforma-tionaboutthepeptideendspresentinlowmassfragments.UsingstepwiseMS,lowmassfragmentionscanalsobedetected(seebelow).Recentlydevelopedoperatingcondi-tionsforiontraps(pulsedQ-dissociation)allowagenerallyenhanceddetectionoflowmassions[41].Hybridinstru-mentssuchastheLTQ-orbitrapcombinationofferanadditionalactivationmodeoccurringinthetransferregionbetweenthetwomassanalyzers,whichgeneratesMS/MSspectrawithhighlyabundantlowmassfragments[42].Currently,MSinaniontrapappearstobethemostapplicabletechniquefordifferentiationbetweenleucineandisoleucine.IntheearlydaysofMS/MS,high-energyCIDwasmorefrequentlyappliedthantoday.Usingthisionactivationmethodforpeptideanalysis,sidechainfrag-mentationswereobserved.Thesewerefoundtobeusefulforthedifferentiationbetweenleucineandisoleucine.PeptidefragmentscontainingLwerefoundtoshowsatel-litesat42Da,comparedto28DasatellitesforI[43].Inlow-energyCIDofpeptidescontainingL/I,theimmoniumionofL/Iat86isfrequentlyobservedwithhighabundance.FurtherMS/MSanalysisofthisimmoniumionshowedthatthefragmentionat69isspeciÞcforI[44Ð46].Iontrapsareparticularlyfavorableforsuchananalysis[41]duetotheirMScapability.SuchananalysisisshowninFig.5providingclearevidencethattheresidueinthepeptideGpSVAVGVIKattheposition2fromtheC-terminusisisoleucine.5MALDI-PSD,MALDI-TOF/TOF,andMALDIin-sourcedecay(MALDI-ISD)MALDI-PSDwasintroducedastheÞrstMALDItechniquefordetectionoffragmentions[47].Fragmentsformedbymetastablefragmentationbetweentheionsourceandthereßectorcanbedetectedinthisway.SubsequentlytheuninterrupteddetectionofacompleteMALDI-PSDspec-trumwasdemonstrated[48],incontrasttotheoriginalstitchingofseveralpartialPSDspectra.MALDI-PSDspectraofpeptidesshowbandyionseriesandneutrallossesasobservedfollowingCID.However,sinceMALDIgeneratesmainlysinglychargedmolecularion,MALDI-PSDspectracontainlesssequenceinformationcomparedtotheCIDspectraofmultiplychargedprecursorionsasgeneratedbyESI.ThefragmentationofMALDIgeneratedpeptideionswasputonanimprovedinstrumentalbasisbythedevel-opmentoftwotypesofMALDI-TOF/TOFinstruments[49,50]allowingtherecordingofpeptideMS/MSspectrawithimprovedsequenceinformation[51,52].Inaddition,severalattemptswereundertakentoimprovethesequenceinfor-mationofMALDI-TOF/TOFspectraofpeptidesbyderiva-tization[53].Mostfrequently,thederivatizationistargetedtothepeptideN-terminuswiththeaimtoenhancetheC-terminalfragmentionseries(yions)whicharerelativelystablespeciesallowingsimpliÞedsequencing[54].Further-morethepKvaluesofthe-aminogroupattheN-terminusandthe-aminogroupoflysineareoftensufÞcientlyapartallowingtheirselectivederivatization.Incontrast,thisisnotfeasiblefortheC-terminus,sincethepKvaluesbetweenC-terminalandsidechaincarboxyfunctionsarelessclearlyseparated.AminogroupspeciÞcmodiÞcationcanbe Table1.TypesofsequenceinformationpresentintheMS/MSspectruminFig.3SNTDANQ[L/I]WT[L/I]KyIonsbIonsNeutrallossFragmentproleCompositionProteasespecicity D A F L G S F L Y E Y S S L D L D S I I A E V L Q G I V S W G S G C A Q A E A E S L Y Q S S Q I D I V L V G G S T F E E L N A D L F D A F L G S F L Y E Y S D A F L G S F L Y E Y S S L D L D S I I A E V S L D L D S I I A E V L Q G I V S W G S G C A Q L Q G I V S W G S G C A Q A E A E S L Y Q S A E A E S L Y Q S S Q I D I V L V G G S T S Q I D I V L V G G S T F E E L N A D L F F E E L N A D L F Figure4.Typicalfragmentionseriesdistri-butioninpeptidesobservedinlineariontrapMS/MSspectra;(A)trypticpeptideswithasinglebasicsite;(B)trypticpeptideswithinternalbasicresidues.Forbothgroupsofpeptides,bandyionserieswithpronouncedoverlapareobserved.J.Seidleretal,634–6492010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.proteomics-journal.com achievedwithsulfonicacidderivatives[55].Forarginine-containingtrypticpeptidesthisleadstoaspeciÞcN-terminalmodiÞcation.Lysine-containingpeptidescanbemodiÞedequallyfollowingguanidylationoflysine-aminogroups[56].Currently,Lysguanidylationisoftencombinedwithderivatizationby4-sulphophenylisothiocyanate(SPITC)[57Ð59]and2-sulfobenzoicacidanhydride[56,60].AnexamplefortheimprovementofaMALDIfragmentionspectrumisgiveninFig.6,adaptedfromarecentdenovosequencingstudyofplantproteinisoforms[61].TheMALDIfragmentionspectrumofthepeptideYVTYAA[I/L]AGDASV[I/L]DDRshowsthesameyfragmentionsbeforeandafterN-terminalderivatizationwithSPITC.However,afterderivatizationbionsarepracticallyabsentandtheyionsareofhighandnearlyuniformabundance.ThesuppressionofbionsbySPITCderivatizationisexplainedbythereplacementoftheN-terminalaminofunctionbythesulfonicacidfunction,sothatthederiva-tizedbfragmentsarenotabletostabilizeanextraproton.AnapproachforinßuencingthefragmentationbehaviorofpeptidesinMALDI-PSDreferstotrypticpeptideswithaC-terminallysine,whichisreactedwithastronglybasicreagent[62,63],resultingintheexclusiveoccurrenceofayionseries.BytheintroductionofdelayedextractionMALDI[64],thedetectionoffragmentionsgeneratedintheMALDIsourcebecamefeasible(ISD)[65].MALDI-ISDdetectsfragmentionsformedinthetimegapbetweenthelaserpulseandtheswitchtothefullacceleratingvoltage(100Ð500ns).ThetypicalMALDI-ISDfragmentionsofpeptidesareoftype,pointingtowardahighabundanceofradicalsintheMALDIplume.ThesametypeoffragmentsareobservedinExDfragmentationtechniques(seebelow),wheretheyoriginatefromprecursorradicalionsgeneratedbyelectrontransfer.MatrixoptimizationforMALDI-ISDhasbeendescribed[66].Applicationsfordatabase-supportedproteinidentiÞcationusingthetopdownapproachhavebeen[67,68]andthetechniqueappearstoofferpotentialalsofordenovosequencing.TheusefulnessofMALDIfordenovosequencingiscurrentlyfurtherstrengthenedbytheavailabilityofMALDI-MS/MSinstru-ments(MALDI-QTOF,MALDI-TOF/TOF,MALDI-6ExDtechniquesRecently,electroncapturedissociation(ECD)[69]andelec-trontransferdissociation(ETD)[70](summarizedasExDtechniques)havebeenintroducedasnewactivationtechni-quesforpeptidefragmentation,whichcanberegardedascomplementarytoCID[71].ExDtechniquesfunctiontransferofasinglelowenergyelectrontoamultiplyprotonatedpeptide,mostlywithchargestate3or4,asoftenobservedinESI.Theelectronistransferredeitherdirectly(ECD)orfromapreviouslyformedradicalanion(ETD).Thefreeradicalsiteintroduceduponelectrontransferleadstoaninstantaneousandlocalradical-inducedbackbonecleavage.ThiscleavageaffectsmainlytheN-Cbond,sothattheresultingfragmentionspectratypicallyshowcorztypeions.Adetailedmechanismfortheirformationhasbeenproposed[70].Recently,thecombina-tionofmetalloendopeptidaseLysNdigestionwithETDorMALDI-TOF/TOFhasbeenappliedfordenovo 30 35 40 45 50 55 60 70 80 85 90 40 60 100 40 60 80 100 40 60 80 100 isoleucine132+86+ 132+86+ 909+86+ BC rel. abundance Figure5.IontrapMS/MSspectraoffordifferentiationbetweenleucineandisoleucine;(A)MS/MSof86generatedfromleucine;(B)MS/MSof86generatedfromisoleucine;(C)MS/MSofgeneratedfromthepeptideGpSVAVGVIK.Thehighabundanceofm/z69in(c)identi-esthepresenceofisoleucine.,634–6492010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.proteomics-journal.com sequencing[72,73].PeptidesgeneratedbyLysNcarryaKresidueattheN-terminusresultinginthepreferentialoccurrenceofcionseries,asituationfacilitatingaread-outofthepeptidesequence.ExDspectraarewellsuitedforsequencingofmodiÞedpeptides[74],sincefragmentationsoriginatingfromsidechainsarenormallynotobserved.EvenrelativelylabilestructuressuchasphosphorylatedaminoacidsstayintactuponExD.However,variationsinthepeptidebackbonestructureinßuencetheradicalinducedfragmentation.Forinstance,cleavageattheN-terminalsiteofPissuppressedduetotheexistenceoftwoN-Cbonds,anobservationthatisinaccordancewiththeproposedfragmentationmechanism.Anotherexampleisthepresenceofanisoaspartylresidue,introducinganextraC-Cbondintothepeptide/proteinbackbone,whichiscleavedusingECD[75,76]orETD[77].ThiscleavageleadstoisoAsp-specifcbackbonefragments.ForisoAspatpositionnfromtheN-terminus,a58ionisformed,andacomplementary57maybeformed,wheremannotatestheisoAsppositioncountedfromtheC-terminus.ArelativequantiÞcationofthesite-speciÞcisoAspcontentinpeptideshasbeendemonstratedbyECD[78].SuchananalysishasalsobeendemonstratedusingCID[79];however,ExDtechniquesappeartobesuperior,sincetheypresentisoAsp-speciÞcions,whereasCIDtech-niquesmainlydisplayquantitativechangesinfragmentionabundances[79]orisoAsp-speciÞcionsofminorrelativeabundance[80].7LCelutionbehavioranddenovoInLC-MS/MSanalyses,thepeptideretentiontimeisananalyticalparameter,whichisobtainedwithoutextraeffort.Nowadays,theLCretentiontimesofpeptidescanbepredictedwithhighreliability(inawindowof2Ð3min)fordifferentchromatographicsystems,[81Ð83].Therelativeelutionorderisalsoausefulparameter,ascanbedemonstratedintheanalysisofthejustmentionedisoAsp-containingpeptides.Sincetheyaregeneratedduringproteinaging,theyaregenerallyaccompaniedbytheircognatescarryinganormalaspartylresidue.OnRP-LC,peptideswithinternalisoAsppeptideselutebeforetheirunmodiÞedanalogs,whereaspeptideswithN-terminalisoaspartateelutelater[84](peptideswithC-terminalisoas- Figure6.MALDIMS/MSspectraofof1H]1ionsofthepeptideYVTYAA[I/L]AGDASV[I/L]DDR(A)beforeand(B)afterSPITCderivatization.Derivatiza-tioninducesneutrallossesofthemodifyinggroup(173Da,215Da)andapronouncedenhancementofallyions.Adaptedfrom[61],withJ.Seidleretal,634–6492010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.proteomics-journal.com partatedonotexist).Inthisway,theLCretentiontimemaybeusefulasanadditionalÔÔsoftÕÕparametertoconÞrmtheresultofadenovosequencingstep.8MassaccuracyandpeptidesequencingThereliabilityofpeptidesequencingimproveswithincreasingaccuracyofthemassmeasurement,sincetheexactmasscontainsinformationabouttheelementalcomposition.ThemostobviousbeneÞtofaccuratemassmeasurementreferstothecorrectassignmentoffragmentionseries.KandQarenominallyisobaricaminoacidswithamassdifferenceofabout36mDa,andthedifferencebetweenWandEGisabout15mDa.Theseambiguitiescanbedifferentiatedatmediumresolution,asprovidedbyQ-TOFinstruments.However,interferingionsofdifferenttypeswithamultitudeofmassdifferencesincludingverysmallvaluesmayoccur,sothathighmassaccuracyisofgeneralvalue.Accuratemassdataarewithoutusefortherecognitionofstructuralisomerssincethesearecharacterizedbyidenticalelementalcompositions.Fordifferentiationbetweenisoele-mentalions,differencesintheirfragmentationbehaviororintheirchemicalproperties,e.g.inderivatizationorlabelexchangereactionshavetobeemployed.Firstofall,thepairL/Icannotbedifferentiatedbymassmeasurement.Inaddi-tion,numerousaccuratemassnumbersexist,whichrepresentdifferentaminoacidcombinations.Forseveraldipeptidecombinations,asingleelementalcompositionmaybeconnectedwithfourstructures(e.g.AN,NA,GQ,andQG).DifferentiationofthesequartetspresentasN-terminalmotifscanbeachievedviathebionfragmentationproÞle[39].OtherexamplesofisoelementalstructuresarethepairsGG/N(bothC)andGA/Q(bothCNowadaysseveralinstrumenttypes(FT-ICR,Orbi-Trap)areavailable[85],providingMSanalysiswithareso-lutionintherangeof100.000Ð500.000withmassaccuraciesintherangeof2Ð0.2ppm.Anearlyexampleforthenewpossibilitiesofferedbysuchextrememassaccuracyistheintroductionofcomposition-baseddenovosequencing[86,87].Thistwo-stepprocedurestartswithcalculationofasetofpossibleaminoacidcompositionsonthebasisofthehighlyaccuratemassvalueforapeptidemolecularionanditsfragmentions.Basedonthecalculationofallmassvalue-compatibleaminoacidcompositions,adatabasecontainingallpermutationsisgenerated.ThisdatabaseisthenusedforassignmentoftheMS/MSspectra.Thenewpossibilitiesofhighlyaccuratemassdataforautomateddenovohavebeensummarizedrecently[88].DenovosequencingandstableisotopeStableisotopelabelingtechniquescanalsobeusedtodenovosequencing.SelectivelabelingoftheC-terminusbyincorporationofOisparticularlyusefulforthispurpose,sinceitisarelativelyfastandcost-effectivemethod.TheÞrstattemptsforOintroductionwereperformedbyacid-catalyzedexchangeandbyesterase-cata-lyzedcleavageofmethylesters[89]withsubsequentanalysisbyfastatombombardment.However,acidtreatmentleadstopronouncedpeptidehydrolysisandesterasecleavageisrelativelyinefÞcientduetoalowafÞnityoftheesterasetowardpeptides.Incontrast,trypticdigestionineffectsafastincorporationofoneortwoOatomsattheC-terminusoftrypticpeptides,asvisualizedbyESIandMALDI-MS[90].Asexpected,aselectivelabelingofyionswasobtainedinthisway.Digestioninamixtureofgeneratesyionswithacharacteristicallydistortedisotopepattern[91Ð93]enablingtheirstraightforwarddifferentiationfromtheunlabeledbions.TheOlabelcanalsobeintroducedafterdigestioninaseparatestep[94].ThecombinationoftrypticdigestionwithOlabelingappearstobeparticularlyuseful,sincetrypticpeptidestendtoshowlonguninterruptedyionseries,andsincebyprinciple,internalyionsdonotoccur.Asanexample,Fig.7showstheMS/MSspectrumofthepeptideSTDANQ[L/I]WT[L/I]K,partiallylabeledwithOatthecarboxyterminus.Allyionscanberecognizedbyanelevated2Dasignalintheirisotopicenvelopes.Differentialterminalderivatizationswithalabeledandanonlabeledreagenthavebeenintroducedbothforquanti-tativeproteomicsaswellasforfacilitateddenovocing.Thelatterpurposeisachieved,sinceN-terminallabelingshiftsonlythebionseries,whereasC-terminallabelingselectivelyaffectsthecorrespondingyions.ThisconcepthasbeendemonstratedforderivatizationwithH-(Nicotinoyloxy)succinimide[95],O-methylisourea[96],-lysine[97],H-formaldehyde[98]ormorerecentlybyN-terminalH-acetylationusingaceticacidanhydrideanhydride10SpecialMS/MStechniquessuitedfordenovopeptidesequencing10.1Multistagefragmentationofsodium-cationizedClear-cutC-terminalsequenceinformationfrompeptidescanbeobtainedbymultiplestageMS[100,101]inaniontrap.Thisisparticularlythecase,whenlithium-orsodium-cationizedpeptidesinsteadofprotonatedpeptidesareselectedasprecursorions.Theprevailingfragmentationprocessof[Mand[MionsistheneutrallossoftheC-terminalaminoacidbuildingblockwithformationofan[Mionofthesametype,butshortenedbyoneaminoacid.Thus,theprocesscanberepeatedusingthecapabilitiesofaniontrap,asdemonstratedinseveralinvestigations.AsequenceladdergeneratedinthiswayisshowninFig.8forthepeptideSQGIASTK.,634–6492010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.proteomics-journal.com Unfortunately,thebroaderapplicationofthiselegantC-terminalsequencingmethodislimitedbythefactthatnomethodsareavailableforthepreferredgenerationofcatio-nizedpeptidesathighsensitivity.10.2Time-delayedfragmentationThedistributionofproductionsobservedfollowingCIDistime-dependent.UsingaQ-Trapinstrument,selectiverecordingofMS/MSspectracontainingpreferentiallythemorestableyions(comparedtobions)hasbeendemon-strated.ThetermÔÔtimedelayedfragmentationÕÕ(TDF)[102,103]hasbeencreatedtodescribethedetectionoffragmentionsproducedatvariabletimewindowsandthussubpo-pulationsofionswithdifferentinternalenergy.ThreemajorstepsareinvolvedinTDF:(i)ionactivation,(ii)ionrelaxa-tion,and(iii)fragmentcollection.TDFprovidestheabilitytosimplifytheproductionspectraasshowninFig.9.IncaseallfragmentionsproducedbyCIDarecollected,theMS/MSspectruminFig.9Aisobtained,showingbothbandyions.Relaxationoftheprecursorionsinthelineariontrapover10msleadstoapopulationofprecursorionswithlowerinternalenergy.SelectiverecordingoffragmentionsoriginatingfromthisrelaxedsubpopulationleadstoasimpliÞedMS/MSspectrumcontainingexclusivelyyionsasshowninFig.9B.Inthisway,TDFsupportsdenovosequencingofpeptides.10.3SingleseriesfragmentionspectraSimpliÞedfragmentionspectraofpeptideswhichexhibitonlyC-terminalorN-terminalfragmentionscanberecor-dedbyusingatwostagefragmentation.Instrumentally,thiscanberealizedusingatriplequadrupoleanalyzerwith Figure7.DenovosequencingbyQ-TOFCIDofthetrypticpeptideSTDANQ[L/I]WT[L/I]K,partiallylabeledwithOatitscarboxyterminus;(A)completespectrum;(B)expandedlowmassregion,demonstratingthefacilediffer-entiationbetweenunlabeledbionsandpartiallyO-labeledy -128.1 K,Q -101.1 T -87.0 S -71.0A -113.1 I,L -75.2 G+H238.0313.2426.3497.3584.3685.4813.5Relative Abundance100[M+Na] -128.1 K,Q -101.1 T -87.0 S -71.0A -113.1 I,L -75.2 G+H238.0313.2426.3497.3584.3685.4813.5Relative Abundance100m / z[M+Na] Figure8.IontrapmultistageMS/MSforC-terminalsequencingofthepeptideSQGIASTKas[Mion.Shownistheoverlayofsixspectra,generatedbyrepeatedfragmentationofthestepwiseshortenedsodiatedpeptideion.J.Seidleretal,634–6492010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.proteomics-journal.com combinedskimmer-CID(sCID)andcollisioncellCID.Thisisapseudo-MSanalysis,sinceskimmerCIDrepresentsafragmentationmodewithoutprecursorionselection.Thus,thisexperimentalsetupwillonlyprovideclearresultsforpuresamplescontainingasinglepeptide.Nevertheless,two-stageCIDforpeptidesequencinghasparticularmerits(Fig.10).ThisisdemonstratedfortheT1fragmentofproteinkinaseA,whichisashortN-terminallymyristoy-latedheptapeptide.Aselectivedetectionofboryionsisachievedusingabionoryion-speciÞcprecursorionscan,respectively.Acommonbionfragmentisfoundatwhichrepresentsthemyristoylfragment,andacommonyionfragmentistheyion.Therefore,usingskimmerCIDincombinationwithprecursorionscanningfor211,onlybionsaredetected,whereasthecombinationwithprecursorionscanningfor147leadstopureyionspectra.Ageneralapplicabilityoftwo-stageCIDfortherecordingofsingleseriespeptideMS/MSspectraasdemonstratedinFig.10requiresthecombinationoftwogenuineMS/MSsteps,ascanberealized,inapenta-quadrupoleinstru-mentwithtwocollisioncells.However,thistypeofinstru-mentiscommerciallynotavailable.11AutomateddenovoEnginesfordenovosequencinghaveundergonecontinuousimprovement[106Ð112]astoolsfordatabase-supportedspectrainterpretation.Basicorfullversionsofseveralofthesetoolsareavailableintheinternet,asmentionedinthecorrespondingpublications.Currently,allresultsprovidedbyautomatednovosequencingshouldbecheckedmanually.Nevertheless,wefoundautomateddenovosequencingveryhelpfulforextractingmeaningfulMS/MSspectrafromlargesetsofMS/MSspectra,thusincreasingthesuccessrateofmanualnovosequencing.Longsequenceionseriesfromcentralpartsofpeptideswereingeneralcorrectlyrecognized,aswellascomplementaryb/yionpairs.Inconsistenciesbetweenproposedandmanualsequenceswereoftenfoundneartheterminalregions,wherecharacteristicdetailsinthespectrawerenotfullyrecognized.Asanexample,Table2showsthreeautomatedannotationsfromadatasetacquiredfordenovosequencingofatypeIIribosome-inactivatingproteinfromX.americana,incomparisonwithmanualsequencingresultsandthelaterdeterminedsequenceofriproximin(Q2PA54).TheresultsinTable2showthattheannotationtoolmayinterchangeasingleaminoacidwithanisobarictwoaminoacidcombination.Thiserroriscausedbythefalse-positiverecognitionofasequenceion.Othererrorsrefertotheannotationofthepeptideends.SincetheCIDspectraingeneralcontainseveralindependentsourcesofinformationforterminalsequences(Table1),thissituationshowsthatthefurtherreÞnementofdenovoannotationtoolstowardabetterrecognitionoftheterminalsequenceisapromisingroutefortheirimprovement.Proteinsoforganismswithunknowngenomeoftenshowsequencehomologiestofunctionallyrelatedhomologsinotheralreadycompletelysequencedorganisms.Therefore,existingsequencedatabasesmaybehelpfulassupportforsequencingresultsobtainedfromunknownproteins.Inpractice,peptidesequencecandidatesmaybesubjectedtohomology-basedsearchprogramssuchasBLAST(basiclocalalignmentsearchtool)ortheFASTA(fastall)algo-rithmtoidentifysimilaritiestosequencespresentindata-bases[113Ð115].Anextensivereviewonhomology-drivenproteomicshasbeengivenrecently[116].Bioinformatictoolsareofhighinterestforfurtheradvancementofdenovopeptidesequencing. Figure9.Q-trapMS/MSspectraofthedoublycharged-caseinfrag-mentat1094(DMPIQAFLLY-QEPVLPGPVR)using(A)conventionalCIDwithfragmentationwindowuptoabout250s,and(B)TDFwithafragmentationwindow10ms.Adaptedfrom[104],withpermission.,634–6492010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.proteomics-journal.com 12CompleteproteinsequencingsupportedbydenovoMS/MSpeptideCompleteproteinsequencemaybeobtainedbyMSalonebytheimplementationofdifferentproteases.Eachproteasegeneratesdifferentsetsofpeptidessothatoverlappingsequencesmaybefoundandlongercontinuoussequencescanbeobtained.Ingeneral,asequenceoverlapofthreeaminoacidsissufÞcientforstitchingoftwosequences,sincemostlyathreeaminoacidsequencemotifisalreadyuniqueforaproteinofintermediatesize(20Ð60kDa).Thecombineduseoftrypsin,chymotrypsinandAspNisbene-Þcialforthispurpose,duetotheirdifferentcleavagechar-acteristicsatbasic,neutral,oracidicsites.Inelastasedigestsnumerousoverlappingpeptidesarefound,duetoitslowspeciÞcity.InspiteofthislackofspeciÞcity,elastasedigestionsleadstopeptidesofaround1kDasize,sincetheafÞnityofelastasetoitssubstratesstronglydecreaseswhenthepeptidelengthfallsbelow0.8Ð1kDa.PeptidesofthislengthcanoftenbesequencedcompletelybyCID,unlessmultiplebasicsitesarepresent.TheÞrstcompleteprimary 454.4383.4596.4268.3 [M+H]+m/z [M+H]+813.5218.2360.3431.3m/z 60-100211 60-100147140840+ESI+ESI280420560700rel. intensity rel. intensity 280420560myrGD A A A A KmyrGD A A A A K [M+H]+ 60-100 myrGD A A A A K Figure10.TriplequadrupolepseudoMSspectraofthepeptidemyrGDAAAAKderivedfromproteinkinaseA;(A)sCIDprecursorscanfor211(myristoylfragmention);(B)precursorionscanfor147(yionofK).ThesescanmodesselectivelyshowsonlythebionseriesA)ortheyionseriesb),respec-tively(seealsoRef.[105]). Table2.Automatedandmanualdenovosequencing(http://www.bioinfor.com/peaksonline)ofpeptidesextractedfromamericanaPrecursorionSequencesTypeofsequence[687.3]2NSADANGA[L/I]EGN[L/I]KProposedSNADANQ[L/I]WN[L/I]KManual[703.9]2TEQQWA[L/I]YPSSPProposedTEQQWA[L/I]YPDRManualAEQKWALFPDRRiproximin(505–515)[695.8]2SNTDANGA[L/I]EGT[L/I]KProposedSNTDANQ[L/I]WT[L/I]KManualSNTDANQLWILKRiproximin(374–385)Somedeviationsbetweenautomatedandmanualsequencingareobserved.Twopeptidesequenceswerefoundtobehomologoustoriproximin(Q2PA54).J.Seidleretal,634–6492010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.proteomics-journal.com structuredeterminationofanentireproteinbyMSalonewasperformedusingmultiproteasedigestionandMS/MS[117].Toourknowledge,thelargestproteinthathasbeenentirelysequencedusingthebottom-upapproachsofarisa21-kDacytochromec[118].Inconnectionwiththebottom-upapproach,twophenomenamaycauseerrorsintheprimarysequence.First,rearrangementprocessesduringiontrapCIDhavebeendescribed[119];second,protease-catalyzedtranspeptidationreactionshavebeenobserved,tothetransferofterminalresiduesortotheligationoforiginallydistantsequenceparts[120,121].Althoughthesephenomenahavebeenclearlydescribed,theiractualimpactontheconÞdenceofdenovoresultsisnotyetexplored.TheintroductionofExDtechniqueshasimprovedthetechnicalbasisforÔÔtop-downÕÕproteinsequencing,fragmentationofcompleteproteins.Forexample,asequencecoverageofabout70%ontheÞrst200residueswasdemonstratedforeachterminusoflargeproteinsgraterthan200kDa[122].Atop-downapproachfortargetedcharacterizationofC-andN-terminiofundigestedproteinsbasedonMALDI-ISDhasalsobeenintroducedunderthesynonymÔÔT-sequencingÕÕ[67].Yooandco-workersrecentlydemonstratedthepotencyofthistechniquebysequencinga31residuepolyethyleneglycolmodiÞedpeptidecompletely[123].ConcerningtheaddressedchallengesandbeneÞtsofbothÐÔÔbottom-upÕÕandÔÔtop-downÕÕÐapproachesitismostlikelythattheywillcontinuetoco-evolveinfutureorwillmeethalfwayashybridapproaches,inwhichlargefrag-mentsorwholedomainsofproteinsareanalyzedintact[124].Peptidedenovosequencingresultscanalsohelptoobtainproteinsequencesmolecularbiologyapproaches.Forinstance,acDNAlibraryisconstructedandprimersderivedfromdenovosequencedpeptidesareusedforRACE-PCR(rapidampliÞcationofcDNA-endswithpolymerasechainreaction)[125,126].FinallythecompleteproteinsequenceisidentiÞedusingDNAsequencing.13ConcludingremarksCIDandETDarecurrentlythemosteffectivetechniquesemployedinpeptidedenovosequencing.BothtechniquesgeneratepeptideMS/MSspectrawithveryhighstructuralinformation.Inthiscontext,CIDisbestsuitedforsmallpeptidesof1Ð2kDa,whereasETDcancopewithlargerpeptides.Inthequestforlargeprecursorions,MALDI-ISDhasshownremarkableprogress.TheresultsofallionizationandfragmentationtechniquesbeneÞtfromtheincreasedmassaccuracyofMS/MSdata,reducingthenumberofsequencescompatiblewithaMS/MSdataset.Incaseofambiguitiesinthedenovosequencingofpeptides,severalchemicalandinstrumentalmethodsexistforimprovingthespeciÞcityoftheresults.However,duetotheirextraefforts,theywillprobablybeappliedinselectivecasesonly.CompletepeptidedenovosequencingbyMS/MSwillnotbegenerallysuccessfulduetointerferingfactors,suchas(i)alowintensitycausingincompletedetectionofsequenceions,(ii)apeptidesequencepreventingtheformationofasufÞcientsetofsequenceions,or(iii)thepresenceofanunusualaminoacidand/oranuncommoncovalentmodiÞcation.Nevertheless,onceacompletepeptidesequencecanbereadfromanMS/MSspectrum,thisresulthasahighlevelofconÞdence.ThisisparticularlythecasewhentheinformationredundancypresentinthemajorityofpeptideMS/MSspectraisusedandwhenadditionaldata,suchasaproteasespeciÞcity,LCelutionbehavior.orfragmentationrules,areintegratedintotheÞnalevaluation.ThefurtherreÞnementofautomateddenovosequencingtoolsinrelationtohighresolutionMS/MSdatamaycomplementthewidelyapplieddatabase-supportedsearchalgorithms.Inthisway,notdatabase-Þledproteinsequencevariations,whichnowremainunassignedinanautomatedannotation,couldalsobereliablyidentiÞed.Thus,inmanyproteomicanalysesthecombinationofdatabase-supportedannotationwithautomateddenovosequencingwillprobablyfurtheradvancetheinterpretationofMS/MSdata.Theauthorshavedeclarednoconßictofinterest.14References[1]Edman,P.,Methodforthedeterminationoftheaminoacidsequenceinpeptides.ActaChem.Scand.,283–293.[2]Fales,H.M.,Nagai,Y.,Milne,G.W.A.,Brewer,H.B.etal.Chemicalionizationmassspectrometryofcomplexmole-cules7useofchemicalionizationmassspectrometryinanalysisofaminoacidphenylthiohydantoinderivativesformedduringEdmandegradationofproteins.,288–299.[3]Schulten,H.R.,Wittmann-Liebold,B.,High-resolutionelddesorptionmass-spectrometry5.Mixturesofamino-acidphenylthiohydantoinsandEdmandegradationproducts.Anal.Biochem.,300–310.[4]Bradley,C.V.,Williams,D.H.,PeptidesequencingusingthecombinationofEdmandegradation,carboxypeptidasedigestionandfastatombombardmentmass-spectro-Biochem.Biophys.Res.Commun.1223–1230.[5]Biemann,K.,Layingthegroundworkforproteomics–Massspectrometryfrom1958to1988.Int.J.MassSpec-,1–7.[6]Biemann,K.,Martin,S.A.,Mass-spectrometricdetermi-nationoftheamino-acid-sequenceofpeptidesandMassSpectrom.Rev.,1–75.[7]Hunt,D.F.,Bone,W.M.,Shabanowitz,J.,Rhodes,J.,Ballard,J.M.,Sequence-analysisofoligopeptidesbysecondaryion-collisionactivateddissociationmass-spec-trometry.Anal.Chem.,1704–1706.[8]Hunt,D.F.,Yates,J.R.,Shabanowitz,J.,Winston,S.,Hauer,C.R.,Proteinsequencingbytandemmass-spec-trometry.Proc.Natl.Acad.Sci.USA,6233–6237.,634–6492010WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.proteomics-journal.com 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