De novo protein sequence analysis of Macaca mulatt

JournalListBMCGenomicsv.8;2007AbstractFullTextPDF(811K)ContentsArchiveRelatedmaterial:PubMedrelatedartsPubMedarticlesby:Tannu,N.Hemby,S.BMCGenomics.2007;8:270.Publishedonline2007August8.doi:10.1186/1471-2164-8-270.Copyright©2007TannuandHemby;licenseeBioMedCentralLtd.DenovoproteinsequenceanalysisofMacacamulattaNileshSTannu1andScottEHemby11DepartmentofPhysiologyandPharmacology,WakeForestUniversitySchoolofMedicine,Winston-Salem,NC,USACorrespondingauthor.NileshSTannu:[e-mail];ScottEHemby:[e-mail]7,2006;AcceptedAugust8,2007.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.TopAbstractBackgroundResultsanddiscussionConclusionMethodsAbbreviationsAuthors'contributionsReferencesAbstractBackgroundMacacamulattaisoneofthemostutilizednon-humanprimatespeciesinbiomedicalresearchofferinguniquebehavioral,neuroanatomical,andneurobiochemcialsimilaritiestohumans.Thismakesitauniqueorganismtomodelvariousdiseasessuchaspsychiatricandneurodegenerativeillnesseswhilealsoprovidinginsightintothecomplexitiesoftheprimatebrain.Amajorobstacleinutilizingrhesusmonkeymodelsforhumandiseaseisthepaucityofproteinannotationsforthisspecies(~42,000proteinannotations)comparedto330,210proteinannotationsforhumans.Thelackofavailableinformationlimitstheuseofrhesusmonkeyforproteomicscalestudieswhichrelyheavilyondatabasesearchesforproteinidentification.WhilecharacterizationofproteinsofinterestfromMacacamulattausingthestandarddatabasesearchengines(e.g.,MASCOT)canbeaccomplished,searchesmustbeperformedusinga'broadspeciesdatabase'whichdoesnotprovideoptimalconfidenceinproteinannotation.Therefore,itbecomesnecessarytodeterminepartialorcompleteaminoacidsequencesusingeithermanualorautomateddenovopeptidesequenceanalysismethods.ResultsTherecentlypopularizedMALDI-TOF-TOFmassspectrometeryieldsacomplexMS/MSfragmentationpatterndifficulttocharacterizebymanualdenovosequencingmethodonaproteomicsscale.Therefore,PEAKSassisteddenovosequencingwasperformedonnucleusaccumbenscytosolicproteinsfromMacacamulatta.Themostabundantpeptidefragments'b-ionsandy-ions',thelessabundantpeptidefragments'a-ions'aswellastheimmoniumionswereutilizedtodevelopconfidentandcompletepeptidesequencesdenovofromMS/MSspectra.Thegeneratedsequenceswereusedtoperformhomologysearchestocharacterizetheproteinidentification.ConclusionThecurrentstudyvalidatesarobustmethodtoconfidentlycharacterizetheproteinsfromanincompletesequencedatabaseofMacacamulatta,usingthePEAKSdenovosequencingsoftware,facilitatingtheuseofthisanimalmodelinvariousneuroproteomicsstudies.TopAbstractBackgroundResultsanddiscussionConclusionMethodsAbbreviationsAuthors'contributionsReferencesBackgroundManyspecieshavebeenusedtomodelvariousaspectsofhumandiseasesincludingmentalillness.However,thecomplexityofhumanbiochemistry,anatomyandbehavioralfactorsarenoteasilymodeledinallspeciesandwarranttheuseofspecieswhichhavegreaterdegreesoffunctionalequivalenceforthemeasuresunderinvestigation.Forexample,theexperimentaluseofMacacamulatta(rhesusmonkey)hasbeenessentialforexpandingourknowledgeofneurodevelopmental,neurodegenerativeandorganichumanbraindiseases,aswellas,fornormalbrainfunctiondueinlargeparttoclosesimilaritiesinneuroanatomy,neurobiochemistryandbehaviorcomparedwithotherspecies.Moreover,theuseofMacacamulattahassignificanttranslationalvalueforunderstandingtheinfluenceofalterationsingeneandproteinexpressioninhumandiseaseprocesses.Onepotentialobstacletocomprehensiveassessmentsofproteinalterationsistherelativepaucityofavailableproteinannotationsforrhesusmonkeys.Currently;NCBInr,Swiss-ProtandTrEMBLlist330210,14991and55805humanproteinannotations,respectively.However,NCBInr,Swiss-ProtandTrEMBLlistonly41968,297and1801proteinannotationsforrhesusmonkey,respectively.Thehighlyuncharacterizednatureoftherhesusmonkeyproteomemakesitdifficulttoidentifyproteins,demonstratedifferentialregulationofproteinsandinvestigatetheirpost-translationalmodifications.Thecharacterizationofproteinsofinterestfromrhesusmonkeyusingthestandarddatabasesearchengines(e.g.,MASCOT)hasalimitationinthat'broadspeciesdatabase'searchesareneededwhichresultsinlessthanoptimalproteinannotation.Thislimitationcanbeovercomeinsomerespectsusingadenovosequencingstrategy,inwhichpartialorcompleteaminoacidsequenceinformationisobtainedusingeithermanualorautomateddenovopeptidesequenceanalysis.ThisapproachhasbeensuccessfullyutilizedinrecentstudiestocharacterizepeptidesboundtoclassIMHCmoleculeHLA-A2.1[1],humanskinelastinprotein[2]andproteinsfromunsequencedgenomeofHalorhodospirahalophila[3].WhilemanualproteinsequencingviaEdmandegradationyieldsexactaminoacidsequencewithoutambiguity,theprocedureislaboriousanddoesnotlenditselftohigh-throughputanalysis.Italsolacksthesensitivityofmassspectrometryandcanbehaltedbythepresenceofblockedaminoacids.Fortunately,automatedsoftwaretoolshavebeendevelopedtocharacterizethe