Serum Biomarkers Identification by Mass Spectrometry in HighMortality Tumors.docx
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Serum Biomarkers Identification by Mass Spectrometry in HighMortality Tumors.docx
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SerumBiomarkersIdentificationbyMassSpectrometryinHighMortalityTumors
SerumBiomarkersIdentificationbyMassSpectrometryinHigh-MortalityTumors
AlessandraTessitore,AgataGaggiano,GermanaCicciarelli,DanielaVerzella,DariaCapece,MariafaustaFischietti,FrancescaZazzeroni,andEdoardoAlesse*
DepartmentofBiotechnologicalandAppliedClinicalSciences,UniversityofL'Aquila,ViaVetoioCoppito2,67100L'Aquila,Italy
*EdoardoAlesse:
Email:
edoardo.alesse@univaq.it
AcademicEditor:
VisithThongboonkerd
Authorinformation►Articlenotes►CopyrightandLicenseinformation►
ReceivedAugust10,2012;RevisedNovember16,2012;AcceptedDecember11,2012.
Copyright©2013AlessandraTessitoreetal.
ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
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Abstract
Canceraffectsmillionsofpeopleworldwide.Tumormortalityissubstantiallyduetodiagnosisatstagesthataretoolatefortherapiestobeeffective.Advancesinscreeningmethodshaveimprovedtheearlydiagnosis,prognosis,andsurvivalforsomecancers.Severalvalidatedbiomarkersarecurrentlyusedtodiagnoseandmonitortheprogressionofcancer,butnoneofthemshowsadequatespecificity,sensitivity,andpredictivevalueforpopulationscreening.So,thereisanurgentneedtoisolatenovelsensitive,specificbiomarkerstodetectthediseaseearlyandimproveprognosis,especiallyinhigh-mortalitytumors.Proteomictechniquesarepowerfultoolstohelpindiagnosisandmonitoringoftreatmentandprogressionofthedisease.Duringthelastdecade,massspectrometryhasassumedakeyroleinmostoftheproteomicanalysesthatarefocusedonidentifyingcancerbiomarkersinhumanserum,makingitpossibletoidentifyandcharacterizeatthemolecularlevelmanyproteinsorpeptidesdifferentiallyexpressed.Inthispaperwesummarizetheresultsofmassspectrometryserumprofilingandbiomarkeridentificationinhighmortalitytumors,suchasovarian,liver,lung,andpancreaticcancer.
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1.Introduction
Cancer-relatedmortalityisoneoftheleadingcausesofdeathworldwide.Themosteffectivetreatmenttofightcancerisstillearlydiagnosis.Ontheotherhand,itisknownthatthecorrectclassificationofthetumor,coupledtoasuitabletherapyandtoastringentfollow-up,helpstopreventanddetectrelapses.Cancerisaveryheterogeneousdisease,and,atthediagnosticlevel,isdefinedbymanyindexessuchashistologicalgrade,tumorstage,patientage,sexand,moreimportantly,geneticbackgroundandprofiles.Histologicalevaluationoftumorspecimensobtainedfromtissuebiopsyisthegoldstandardofdiagnosis,butoftentumorswiththesamehistopathologicalfeaturesresponddifferentlytothesametherapy.Newgenerationdiagnosticplatforms,previouslyunavailable,haveenabledtobettercharacterizetranscriptomicsignaturesthatpredicttumorbehaviour,helpingtodefinediagnosis,prognosis,andthemostappropriatetherapies[1–3].Tumorbiomarkerdiscoveryinbiologicalfluids,suchasserum,plasma,andurine,isoneofthemostchallengingaspectsofproteomicresearch[4].Manyresearchershaveattemptedtoidentifybiomarkersinserumthatreflectaparticularpathophysiologicalstate.Sincetheexpressedproteins,native,fragmented,orposttranslationallymodified,quicklychangeinresponsetoenvironmentalorpathologicalstimuli,theserumproteomeisconsidereddynamic,oppositelytothestablenatureofthegenome.Proteinsandtheirfunctionscandeterminethephenotypicdiversitythatarisesfromasetofcommongenes.Thestudyoftheserumproteomehighlightsdifferencesinproteinexpressionreflectingaspecificpathologicalstateandprovidesusefulinformationtodiagnoseadisease,toevaluateprognosisortherapyresponse[5].
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2.BiomarkerDiscoveryinCancer:
TheComplexityofHumanSerum
Singleorasmallnumberofserum-basedbiomarkersindicativeofcancerprogression,suchasprostate-specificantigen(PSA),alpha-fetoprotein(AFP),CA-125,CA-15.3,CA-19-9,orCEAforprostate,liver,ovary,breast,pancreas,orcoloncancer,arecurrentlyused.Mostofthesemoleculeshavebeenisolatedfromanimalsimmunizedwithtumorcellsextractsorcelllines,withsubsequentscreeningformonoclonalantibodiesagainstcancer-associatedantigens[6].Theabove-mentionedproteinsincreasetheaccuracyofdiagnosis,eventhoughthereisanurgentneedtoisolateanduseinclinicalpracticemorespecificbiomarkers,orgroupsofbiomarkers,topreciselycharacterizethediseaseatthediagnosticorprognosticlevelandtomonitoritsprogression[7,8].Biomarkerscouldalsohelptopredicttheresponseofthepatienttoanticancertherapyandtherebytoguidephysiciansinchoosingthebesttreatment.Thisresearchismoreappealingduetothesimplicityofobtainingbloodsamples,but,atthesametime,showslimitsduetothecomplexityoftheserumproteinmixtures.Aplethoraofmoleculesfromalmosteverytissueofthebodycanbefoundinhumanserum/plasma.Manyoftheserumproteinsarepresentatverylowconcentrations(lessthanpg/mL),whileothersarepresentinverylargeamounts(morethanmg/mL).Serumandplasmaareverycomplexmixturesofproteinsandexhibitabroaddynamicrangeofrelativeabundance(upto12ordersofmagnitude)[9].Theycontainthousandsofproteins,whosesomeareveryabundant(e.g.,albumin,immunoglobulins,apolipoproteins)andconstituteapproximatelythe95%ofthetotalproteincontent,butonlythe0.1%oftotalproteinspecies[10,11].Forthesereasons,itisthoughtthatmanypotentiallyimportantproteinsandmarkers,ifpresentatlowconcentrations,canescapethedetection.Evidencesshowthatthecirculatingfragmentsfromunmodifiedorpost-translationallymodifiedproteinsgeneratedinthetumortissuemicroenvironmentcanbeusedasdiagnosticorprognosticmarkers.Proteolysiswithinthetissueorderegulatedpost-translationalevents(e.g.phosphorylation)generateproteinfragmentsthatdiffuseintothecirculationandcouldgiveinformationaboutthepresenceortheprogressionofthedisease,thenfacilitatingthemanagementofthetumor.Amongthesefragments,thefractionwithlowmolecularweight,thepeptidome(<20
KDa,LMWpeptides),isprotectedfromrenalclearancebyinteractionwithabundantserumproteinsand,inparticular,seemstobeanimportantsourceofbiomarkers[12].
Inordertosimplifythebiomarkerdiscoveryprocess,manyprefractionationmethodshavebeendevelopedtoremovehighlyabundantproteins.Theseusefulprecursorsofproteomicanalysisincludetheuseofimmobilizeddyes(cibacronblue)[13,14],immunoaffinity-basedtechniques[15,16],solidphasefractionation[17],liquidchromatography[18],orlow-molecularweightfractionenrichment[19–21].ApromisingmethodunderinvestigationisbasedontheuseofN-isopropylacrylamide(NIPA)nanoparticleswhichallowafastone-stepcaptureandconcentrationofanalyteslessthan20–25
KDainmolecularweight[22–24].Atthesametime,thenanoparticlesareabletoprotecttheproteinsfromthedegradationduetothe“exvivo”enzymaticactivityofserumproteases,and,whenconjugatedwithsuitablechemicalbaits,showhighercapabilitytosequesterandretainmanydifferentproteinsfromwholeserum,onthebasisoftheirchemicalandphysicalproperties[25].Withthismethod,thecapturedanalytescanberecoveredbyasimpleelectroelutionandthenanalyzedbyHPLC-MS/MS,westernblottingorimmunoassaysforcompletemolecularcharacterization.Toreducethecomplexityofserumproteome,severalstudiesarefocusedontargetingaspecificsubsetofserumproteins[26].Glycosylationisoneofthemostcommonposttranslationalmodificationsinproteins.Approximately50%ofknowneukaryoticproteinsareglycosylated[27].Itisknownthatcancercellsexpressaberrantglycosylationpatterns[28].Theanalysisofglycosylatedproteins(glycoproteome)hasreceivedgreatinterest,becauseoftheglycoproteicnatureofthecurrentlyusedcancerbiomarkers.Twomajormethodshavebeendevelopedtoenrichglycoproteinsorglycopeptides,basedonchemicalcapture(reactionbetweenaldehydegroupsandhydrazide)[29,30]andlectin-affinitycapture(specificrecognitionofproteinglycanmoietiesbylectins)[31].
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3.MassSpectrometry:
TheCancerBiomarkerDiscoveryTool
Manystudieshavebeenfocusedontheidentificationofnewserumbiomarkersbymassspectrometry(MS).Thispowerfulmethodenablestoidentifyaproteinwithoutrequiringtheknowledgeofitsaminoacidsequence.Furtherimprovementofthistechnologyhasprovidedhighaccuracytodefinemass-to-chargeratio(m/z)andtogeneratehigh-resolutionspectra.Inaddition,thedevelopmentoftandemmassspectrometry(MS/MS),abletoprovidedenovoproteinsequenceinformation,hasenhancedtheapplicationsofthistechnologyinproteomics[32,33].SeveralMSmethodshavebeenusedtocharacterizebodyfluids.Differentcombinationsofionizationsources(e.g.,MALDI,ESI),analysers(e.g.,timeofflightTOF,quadrupole,Fouriertransformandquadrupoleiontraps),andfragmentationmethods(e.g.,CIDcollisioninduceddissociation,ETDelectrontransferdissociation)canbeused.MALDI-TOF(Matrix-AssistedLaserDesorption/Ionization-timeofflight)[34]isbasedonasoftionizationmethodwherealaserbeamgeneratesevaporationofacrystallizedsample-matrixmixture.MALDIisusedinbiochemicalareasfortheanalysisofproteins,peptidesandoligonucleotides.Surface-EnhancedLaserDesorption/IonizationTime-of-Flight(SELDI-TOF),amodificationofMALDI-TOF
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