Mass spectrometry-based methods for protein identification and 5基于质谱的蛋白质鉴定和方法

MS-basedmethodsforproteinidentification&phosphorylationsiteanalysis

생명과학부구용의MassSpectrometer(MS)분자가MS내로들어가면분자는이온화됨과동시에더작은이온들(fragments)로쪼개진다.쪼개진이온들은그들의질량/하전(m/z)비에따라선택적으로분리되어이온수에비례하게signal을만든다.이온들의질량/하전비는이온들의생성된양(Abundance)의함수로표시되어massspectrum이그려지며,이massspectrum을이용하여미지성분의정성확인을할수있다.이온은양이온과음이온모두사용MSComponentMScomponentSampleinlet시료도입장치로시료를MS내로효율적으로보내주는역할을한다Ionsource시료분자를이온화시키고더작은이온으로쪼갠다.생성된이온들을MSanalyzer쪽으로이동시킨다Massanalyzer이온들을m/zratio에따라선택적으로분리시킨다Iondetector이온흐름을그양에비례하게전기적인흐름으로전환,증폭시켜signal을생성한다Vacuumsystem

MS내진공상태를10-4~10-9Torr로만들어주어최적의상태로분석이진행될수있도록한다DataSystem

MS내각구성성분들의조절이가능하며시료분석과동시에데이터해석을할수있는곳이다Ionsource

Gaseoussampleintroduction

-EI(electronionization)-CI(chemicalIonization)Liquidsampleintroduction-FAB(fastatombombardment)-ESI(electrosprayionization)(softionization)

Solidsampleintroduction

-MALDI(softionization)(matrix-assistedlaserdesorption/ionization)

EI(electronionization)전자를발생시키기위한filament가가열되고(+)극판에전압이걸리면가속된전자들이흐르고그속을지나던기체화된sample은전자와충돌하여에너지를얻고전자하나를잃어분자이온M+가된다이방법은큰에너지를사용하기때문에복잡한spectrum을이루며분자이온을얻기힘들다M+e-

⇒M++2e-CI(chemicalIonization)가열된filament에서발생,가속된전자는106정도로많은reagentgas와충돌하여이들을이온화시키고이reagentgasion은samplegas와충돌이samplegas는fragmentation되기도하고때로는reagentgasion과complex를이루기도한다.매우낮은에너지로충돌하기때문에EI보다수백배이상많은수의분자이온을만들어내기때문에분자량확인에많이쓰인다.R(CH4)+e-

⇒R++2

e-

R++M⇒M1++N1M1+⇒M2++N2

MALDI

MALDIprocessmatrix와sample을각1000~10,000:1의농도비율로적당한용매(acidicorganicsolvent-TFA+MeCN)에혼합한다이혼합물을시료용probe에올려놓고진공조건을만들어주면유기용매는기화되고시료는matrix와함께균질하게결정화된다이때펄스레이져(UVat337nm,IRat2.94um)를sample-matrix결정에쏘아주면에너지가matrix를통해sample로전달되어약한이온화가일어난다.이온화는protonation/deprotonation,cationattachment/cationdetachment,oxidation/reduction으로일어난다.MatrixinMALDISinapinicacid:peptide,protein>2kDa2,5-Dihydroxybenzoicacid:glycoprotein,glycolipids,carbohydrateA-Cyano-4-hydroxycinnamicacid:lowMWpeptides,peptide2-Benzoicacid:sulfonateddyesNicotinicacid/Anthranilicacid(1;1):oligonucleotide,sialylatedglycopeptides3-Hydroxypicolinicacid:oligonucleotideadducts,oligonucleotide2,4(6)Trihydroxyacetophenone:oligonucleotide,proteins1-30kDaESIESI(electrosprayionization)시료용액이고전압이걸려있는capillary를통과하면서분무되어전하를많이띤droplet이생성됨drpolet이capillary에서orifice를지나면서inertgas(orheat)에의해desolvation됨Desolvation과정에서ion의charge는더증가하고“Coulombicexpolsion”에의해droplet의ion이gasphase로된다.smaple에가해지는충격이약하며,multiplecharge를가진peptide이온이생긴다.ESIMassanalyzerandDetectorMALDI–MS(Linear)TOF(timeofflight)Ionsource에서생성된이온을높은전압으로가속화시켜tube(field-freedrifttube)를통과시켜검출기에도달하게한다.모든이온들이ionsource에서같이떠난다면m/z가작을수록더빨리검출기에도착한다.TOF는이온들의이동시간을측정하여m/z를계산한다TOFMassresolutionResolution=m/△mMALDI-TOFMSresolution

Reflectron(ionmirror)-비행관끝에위치한일련의정전기장과자기장으로이루어있음-m/z값은같으나약간씩다른운동에너지를가지고있는이온들은reflectron에가까이도달할수록속도가늦어져정지하여이온들의속도차가줄어들고reflectron에서반사되어detector쪽으로비행한다Time-lagfocusing(delayedextraction)-이온의생성과가속사이에약간의시간차를주어이온들의초기운동속도분포차를줄여주는방법MALDI–MS(Reflectron)Quadrupleanalyzer(massfilter)

4개의molybdenum막대로이루어져있으며,한쌍(1,2)은dcvoltage가또다른한쌍(3,4)은radiofrequencyvoltage가가해진다.Dcvoltage가0이고RFvoltage만있으면모든이온이통과할수있다가해지는전압의진폭은선택된m/z의비에해당되는ion만ionsource에서detector까지통과하도록한다이quadropole의전압을바꾸면서주어진mass범위의이온을scanning하여massspectrum을얻는다.PSD(postsourcedecay)ThemetastablefragmentationofionsafterfullaccelerationthatoccursinthefieldfreeregionofTOF-MSIfpeptidesaresubjectedtoPSDtheywillfragmentpredominantlyalongthpolypeptidebackbone,thusgeneratingseriesoffragmentionswhich,inprinciple,containtheaminoacidsequenceinformationofthepeptideToobtainprimarystructuralinformationMALDI–MS(Reflectron)MS/MSESI–TQ-MSESI–TQ-MSCID(collision-induceddissociation)ThetermusedtodescribefragmentationinMS/MSexperiment.Theprecursorionisisolatedandallowedtocollidewithneutralgasmoleculesinacollisioncell.Thetranslationalenergyoftheprecursorionisconvertedtointernalenergyafterthecollisionsresultinginfragmentationoftheprecursorion.quadruple을사용하는방법에따라여러가지mode로사용할수있음-MSmode-MS/MSmode-Neutrallossscanmode-Precusor(orparent)ionscanning-In-sourceCID:fragmentationoccursinthehigh-pressureregionofanESIsourceasaresultofcollisionwithatmosphericgases.Ringelectrode와end-capelectrode로구성되어있으며,quadruple처럼dcvoltage와RFvoltage가흐른다.RFonly인경우모든이온들이trap에갇히게됨ESI–IT-MSESI–IT-MSTandemmassspectrometerinwhichionscanbeaccumulatedandstoredpriortoanalysisTheiontrapisbothamassanalyzerandcollisioncell“Resonanceejection”referstoionsbecomingunstableinthetrapandbeingejectedaxiallythroughthend-capelectrodeswheretheyaredetectedThroughthisprocessoftrappingandselectiveejectionofions,ionsofspecificm/zcanbeisolatedinthetrapMassspectrumofCO2TotalionchromatogramEI-MSspectrumofpropionicacidPADatabaseExperimentaldataPropionicacidM+MALDI-MSspectrumofproteinMS/MSspectrumofpeptidePeptidemasssearchingPeptidesaregeneratedbydigestionoftheproteinofinterestusingspecificcleavagereagents(usuallyenzymes)ThemassesofthesepeptidesareaccuratelydeterminedexperimentallyusingMALDI-MS(orESI-MS)Theoreticalpeptidemassesarecalculatedforeachsequenceentryinthedatabaseusingthesamecleavagespecificityasthereagentemployedexperimentally.Ascore(orranking)isthencalculatedtoprovideameasureoffitbetweentheexperimentallyderivedandcalculatedpeptidemasses.Measuredpeptidemass와sequence가맞지않는경우Theadditionalmassesareduetoposttranslationalorartifactualmodificationsorpost-translationalprocessingUnspecificproteolysishadoccurredorcontaminatingproteasewaspresentProteinwaspartofamixtureof‘contaminating’proteinsCriticalexperimentalparameter

inpeptidemasssearching

Theaccuracyofthepeptide-massmeasurement-time-lagfocusing/delayedextraction-internalstandardwithisotopeThespecificityoftheenzyme(orchemicalreagent)employed-‘missedcleavage’,‘raggedtermini’를control할수있도록program화OrthogonalmethodinpeptidemasssearchingSite-specificchemicalmodificationDeterminationofpartialaminoacidcompositionofthepeptideIdentificationoftheN-terminalaminoacidresidueIdentificationofdifferentcleavagesiteswithinthepeptidesIdentificationoftheC-terminalresidue(s)Fragmention“bion”-thefragmentwithc-terminaldeletionsandintactN-terminal“yion”-thefragmentwithN-terminaldeletionsandintactC-terminalinternalfragment-internalacylion-immoniumion(representindividualaminoacid)UninterpretedfragmentionsearchingFragmentationcanbeinducedbyPSD-MALDI-MSaswellasbyCIDintriplequadrupleorion-trapmassspectrometerFragmentionspectracontainreduntantpiecesofinformationHowtointerpretfragmentionManualinterpretationInterpretwithdatabase-Apartialmanualinterpretationofthespectrumtoidentifyconsecutiveelementsofaparticular(bory)ionseries-Uninterpretedfragmentionsearchprogram(SEQUEST)

DenovosequencingDatabaseindependent

“peptideladdersequencing”-differentpeptideinlengthbyoneaminoacid-bychemical(Edman)⇒N-terminalblocking:Gln(128.13),Lys(128.17)구별,Ile,Leu구별못함-enzymaticdegradation:IleandLeu,GlnandLys구별못함-areanalyzedbyMALDI-TOFMSCIDspectra(fragmentionspectra)

-aremanuallyinterpretated-missingfrgament(incompleteionseries)확인을위해trypsin분해시H218O사용(50%H218O+50%H216O,intactC-terminalpeptideionserieshavedoubletby2u)-methylesterficationofthecarboxylgroupsinthepeptide(14uincrease,derivatizedandunderivatized비교)PeptideladdersequencePhosphorylationsiteanalysisstrategies

Complicationofphosphoproteinanalysis-thefrequentlylowstoichiometryofphosphorylation-thepresenceofmultiple,differentiallyphosphorylatedformsInvitroanalysis-scaleupofproteinbykinasereaction-comparisonwith2D-PPmapsofinvivo(confirmationofidentityindirectly)-MSanalysisDetectionandisolationofphosphoproteinsFortheanalysisofthesite(s)ofproteinphosphorylation-purificationofphosphoprotein-enzymaticorchemicalfragmentationofthephosphoprotein-Isolation,separation,analysisofpeptideIsolation

-separationofproteinsbygelelectrophoresis-fragmentationofthephosphoproteinbandorspot-extractionofthegeneratedphosphopeptideMorepositiveidentification-32Pradiolabelling:invivo(32PO4),invitro([γ-32P]ATP)-westernblotting:particularlytyrosinephosphorylatedproteinSeparationofphosphopeptides필요한이유-농도를농축하는역할을하여S/N비를높임-radiolabel의activity를이용하여phosphopeptide의상대적또는절대적인양을구할수있음-separation에의해확보된재현성으로단백질의phosphorylation상태를정량적으로결정할수있음-nonpeptidecontaminants를제거하여적은양의phosphopeptide의분석을용이하게함PhosphopeptideseparationtechniquesBy2-dimensionalphosphopeptidemapReversed-phaseHPLCHigh-resolutiongelelectrophoresisImmobilizedmetalaffinitychromatogrphy(IMAC)Phosphopeptide는상대적,절대적으로적은양때문에분석이어려우므로이러한점을극복할수있는최적의separation방법을선택해야Separationby2D-PP1stdimensionbyelectrophoresisonthin-layercelluloseplate+2nddimensionbyTLConthesameplateinformation-radiolabelledspot수⇒phosphorylatedsites최대수-radiolabelledspot의intensity

⇒peptide들의상대적인phosphorylation정도-relativestateofhydropathybetweenphosphopeptieMSanalysisafterextractionfromplate-protease양이중요sensitiveandreproducibilebyradiolabellingSeparationbyRP-HPLCReproducibleandsimplecolumn으로분리하고radioactivitycount로fraction

⇒count를시간의함수로하여radioactivefraction의수를알수있음단점-veryhydrophilicphosphopeptide,veryhydrophobicphosphopeptide의분리가어렵다-2D-PP보다resolution이낮다-phosphopeptidewillsticktometalsurface장점-ESIMS와online으로연결하여사용할수있다(LC-MS/MS)-isotope을사용할수없는인체단백질분석가능Separationbyhigh-resolutionelectrophorsisandIMAC

High-resolutiongelelectrophoresis-2-DE-특정phosphopeptide의손실이많지만널리보급되어있어사용하기좋음IMAC-같은sequence를갖는nonphosphorylatedpeptide에비하여상대적으로매우적은양의phosphorylatedpeptide의분석어려움-separationandenrichment1)phosphopeptide와metal(Fe3+,Ga3+)의chelating2)elutionbyphosphateorincreasedpH3)acidicaminoacid도enrichment되는단점Detreminationofthetypeofphosphorylatedaminoacid이유가능한phosphorylatedsite의수를찾아냄으로써polypeptide내의phosphorylatedresidues의assignment를쉽게할수있음Technique1)phosphoaminoacidanalysis-32P-aminoacid(hydrolysateof32P-labeledphosphoproteinorphosphopeptide)⇒autoradiography-phosphoaminoacidstandard⇒ninhydrinstaining-sample과standard의비교분석(보통1site/phosphopeptide)2)phosphoaminoacid-specificimmunodetction

-antibodiesspecificforparticularphosphoaminoacid

-상업적으로antibody판매

Determinationof

thesiteofphosphorylationChemicalphosphopeptidesequencing

-phosphopeptidesequencingbystep-wisechemicaldegradation(nonradioactive,radioactivemethods)-analyzedasphenylthiohydantoylderivatives-notavailableinverylimitedamountMassspectrometricanalysisofphosphopeptides

-phosphopeptide의양이1pmole이상이면2D-PPmap에서extraction이가능하고,MS로분석이가능-twobasictheme1)chemicallabilityofthephosphateesterbonds2)thedetectionofthemassaddedtoapeptide(80u)-productionscaninatandemMS으로phosphorylationsite확인

⇒phosphorylatedaminoacidtype을알고있으면더용이MassscanforphosphopeptidesanalysisIn-sourceCID-identifyphosphopeptidesbyobservationofH2PO4-(97U),PO3-(79U)andPO2-(63U)-detectphosphopeptidesinnegativeionmodeandthenswitchtopositiveionmodeNeutrallossscan-positiveionmodewithESIinaTQMS-Q1,Q3arescannedoverdifferentm/zranges-neutrallossofphosphoserineandphosphothreonine:98Mass