a comprehensive resource for integrating and displaying protein ptms

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A comprehensive resource for integrating and displaying proteinpost-translational modifications

BMC Research Notes 2009, 2:111 doi:10.1186/1756-0500-2-111

Tzong-Yi Lee ([email protected])Justin Bo-Kai Hsu ([email protected])

Wen-Chi Chang ([email protected])Ting-Yuan Wang ([email protected])

Po-Chiang Hsu ([email protected])Hsien-Da Huang ([email protected])

ISSN 1756-0500

Article type Data Note

Submission date 18 November 2008

Acceptance date 23 June 2009

Publication date 23 June 2009

Article URL http://www.biomedcentral.com/1756-0500/2/111

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Acomprehensiveresourceforintegratinganddisplayingprotein

post-translationalmodifications

Tzong-YiLee1,JustinBo-KaiHsu1,Wen-ChiChang1,Ting-YuanWang1,Po-ChiangHsu2,andHsien-DaHuang

1,3,4,

1DepartmentofBiologicalScienceandTechnology,InstituteofBioinformaticsandSystems

Biology,NationalChiaoTungUniversity,Hsin-Chu300,Taiwan2DepartmentofBiologicalScienceandTechnology,InstituteofBiochemicalEngineering,

NationalChiaoTungUniversity,Hsin-Chu300,Taiwan3DepartmentofBiologicalScienceandTechnology,NationalChiaoTungUniversity,

Hsin-Chu300,Taiwan4CoreFacilityforStructuralBioinformatics,NationalChiaoTungUniversity,Hsin-Chu300,

Taiwan

Correspondingauthor

Emailaddresses:

TYL: [email protected]

JBKH:[email protected]

WCC: [email protected]

TYW: [email protected]

PCH: [email protected]

HDH: [email protected]


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Abstract

Background

ProteinPost-TranslationalModification (PTM)playsanessentialrole incellularcontrolmechanisms thatadjust

protein physicalandchemicalproperties,folding, conformation, stabilityandactivity, thusalsoaltering protein

function.

Findings

dbPTM (version 1.0), which was developed previously, aimed on a comprehensive collection of protein

post-translational modifications. In thisupdate version (dbPTM2.0),wedevelopeda PTMdatabase towards an

expertsystemofproteinpost-translationalmodifications.Thedatabasecomprehensivelycollectsexperimentaland

predictiveproteinPTMsites.Inaddition,dbPTM2.0wasextendedtoa knowledgebasecomprisingthemodified

sites,solventaccessibilityofsubstrate,proteinsecondaryandtertiarystructures,proteindomains,proteinintrinsic

disorderregion,andproteinvariations.Moreover,thisworkcompilesabenchmarktoconstructevaluationdatasets

forcomputationalstudytoidentifyingPTMsites,suchasphosphorylatedsites,glycosylatedsites,acetylatedsites

andmethylatedsites.

Conclusions

The current release not only provides the sequence-based information, but also annotates the structure-based

informationforproteinpost-translationalmodification.Theinterfaceisalsodesignedtofacilitatetheaccesstothe

resource. Thiseffectivedatabaseisnowfreelyaccessibleathttp://dbPTM.mbc.nctu.edu.tw/.


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Findings

Background

Protein Post-Translational Modification (PTM) plays a critical role in cellular control mechanism, including

phosphorylation for signal transduction, attachment of fatty acids for membrane anchoring and association,

glycosylation for changing protein half-life, targeting substrates, and promoting cell-cell and cell-matrix

interactions, and acetylation and methylation of histone for gene regulation [1]. Several databases collecting

information about protein modifications have been established through high-throughput mass spectrometry in

proteomics. UniProtKB/Swiss-Prot [2] collects many protein modification information with annotation and

structure. Phospho.ELM [3], PhosphoSite [4] and Phosphorylation Site Database [5] were developed for

accumulatingexperimentallyverifiedphosphorylationsites.PHOSIDA[6]integratesthousandsofhigh-confidence

invivophosphorylationsitesidentifiedbymassspectrometry-basedproteomicsinvariousspecies.Phospho3D[7]

isadatabaseof3Dstructuresofphosphorylationsites,whichstoresinformationretrievedfromthephospho.ELM

databaseandisenrichedwithstructuralinformationandannotationsattheresiduelevel.O-GLYCBASE[8]isa

databaseofglycoproteins,mostofwhichincludeexperimentallyverifiedO-linkedglycosylationsites.UbiProt[9]

stores experimental ubiquitylated proteins and ubiquitylation sites, which are implicated in protein degradation

throughanintracellularATP-dependentproteolyticsystem.Moreover,theRESIDproteinmodificationdatabaseisa

comprehensivecollectionofannotationsandstructuresforproteinmodificationsandcross-links,includingpre-,co-,

andpost-translationalmodifications[10].

dbPTM [11] was developed previously to integrate several databases to accumulate known protein

modifications,aswellastheputativeproteinmodificationspredictedbyaseriesofaccuratelycomputationaltools

[12,13].ThisupdatedversionofdbPTMwasenhancedtobecomeaknowledgebaseforproteinpost-translational

modifications,whichcomprises a variety of new features including the modified sites, solvent accessibility of

substrate, protein secondary and tertiary structures, protein domains and protein variations. We also collected

literature related to PTM, protein conservations and the specificity of substrate site. Especially for protein

phosphorylation,thesite-specificinteractionsbetweencatalytickinasesandsubstratesareprovided.Furthermore,a

variety of prediction tools have been developed for more than ten PTM types [14], such as phosphorylation,

glycosylation,acetylation,methylation,sulfationandsumoylation.Thisworkconstructedabenchmarkdatasetfor

computationalstudiesofproteinpost-translationalmodification.Thebenchmarkdatasetcanprovideastandardfor


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measuring the performance of prediction tools that have been presented for identifying post-translational

modificationsitesofproteins.ThewebinterfaceofdbPTMisalsoredesignedandenhancedtofacilitatetheaccess

totheproposedresource.

Dataconstructionandcontent

As shown in Figure 1, the system architecture of dbPTM2.0 database comprises threemajor components: the

integrationofexternalPTMdatabases,thecomputationalidentificationofPTMs,andthestructuralandfunctional

annotations of PTMs. We integrated five PTM databases, including UniProtKB/Swiss-Prot (release 55.0) [1],

Phospho.ELM (version 7.0) [15], O-GLYCBASE (version 6.0) [8], UbiProt (version 1.0) [9] and PHOSIDA

(version 1.0) [6] for obtaining experimental protein modifications. The description and data statistics of these

databasesarebrieflygiveninTableS1(seeAdditionalfile1 -TableS1).Additionally,HumanProteinReference

Database(HPRD)[16],whichcompilesinvaluableinformationrelevanttofunctionsandPTMsofhumanproteins

inhealthanddisease,wasalsointegrated.

In the part ofcomputational identificationofPTMs,KinasePhos-like method [11-13,17]wasapplied for

identifying20typesofPTM,whichcontainatleast30experimentallyverifiedPTMsites.Thedetailedprocessing

flowofKinasePhos-likemethodsisdisplayedinFigureS1(SeeAdditionalfile1-FigureS1).Thelearnedmodels

were evaluated using k-fold cross validation. Table S2 (See Additional file 1 - Table S2) lists the predictive

performanceofthesemodels.Toreducethenumberoffalsepositivepredictions,thepredictiveparameterswereset

toensureamaximalofpredictivespecificity.

ThestatisticsoftheexperimentalPTMsitesandputativePTMsitesinthisintegralPTMdatabaseisgivenin

Table1.AfterremovingtheredundantPTMsitesamongsixdatabases,therearetotally45833experimentalPTM

sitesin thisupdateversion.AllexperimentalPTMsitesarefurthercategorizedbyPTMtypes.For instance,there

are31,363experimentalphosphorylationsitesand2,080experimentalacetylationsitesinthedatabase.Inaddition

totheexperimentalPTMsites,UniProtKB/Swiss-ProtprovidesputativePTMsitesbyusingsequencesimilarityor

evolutionary potential. Moreover, KinasePhos-like methods [11-13, 17] were adopted to construct the profile

hiddenMarkovmodels(HMMs) for twentytypes ofPTMs.Thesemodelswereappliedto identifythe potential

PTMsitesagainstproteinsequencesobtainedfromUniProtKB/Swiss-Prot.AsgiveninTable1,2,560,047sitesfor

allPTMtypeswere identified.The structural andfunctionalannotations ofproteinmodificationswereobtained

fromUniProtKB/Swiss-Prot[18],InterPro[19],ProteinDataBank[20]andRESID[10](SeeAdditionalfile1-

TableS3).


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Utilityandmajorimprovements

Inorder toprovidemore effective informationaboutprotein modifications inthisupdate version,weextended

dbPTMto aknowledge base containing structuralproperties forPTM sites,PTMrelated literature,evolutionary

conservationofPTMsites,subcellularlocalizationofmodifiedproteinsand thebenchmarksetfor computational

studies.Table2showstheenhancementandnewfeaturessupportedinthisstudy.Firstofall,theintegratedPTM

resourceismorecomprehensivethanpreviousdbPTM,whichenrichesthePTMtypes,varyingfrom373to431

PTMtypes.TodetectthepotentialPTMsitesinUniProtKB/Swiss-ProtproteinswithoutanyPTMannotations,the

KinasePhos-like method was applied to 20 PTM types. Especially in protein phosphorylation, more than 60

kinase-specificpredictionmodelswereconstructedandappliedtoidentifythephosphorylationsiteswithcatalytic

kinases.

StructuralpropertiesofPTMsites

In order to facilitate the investigation of structural characteristics surrounding the PTM sites, protein tertiary

structure obtained from ProteinDataBank [20] was graphicallypresentedbyJmolprogram.Forproteinswith

tertiary structures (5% of UniProtKB/Swiss-Prot proteins), the protein structural properties, such as solvent

accessibility and secondary structure of residues, were calculated by DSSP [21]. The solvent accessibility of

residuesandsecondarystructureofresiduesforproteinswithouttertiarystructureswerepredictedbyRVP-net[22]

andPSIPRED[23],respectively.TheintrinsicdisorderregionswereprovidedusingDisopred2[24].

Figure 2 depicts an illustrative example that Insulin Receptor Substrate 1 (IRS1) of human

(UniProtKB/Swiss-ProtID: IRS1_HUMAN)can interactwithInsulinReceptor (INSR)andinvolveintheinsulin

signaling pathway [25]. Three fragments of ISR1 protein have tertiary structures inPDB. Structure 1K3A the

protein region from 891AA to902AA.Two experimental phosphorylation sites S892 andY896 locate inthe

region, and their solvent accessibility and secondary structure can be derived from the tertiary structures.The

solventaccessibilityandsecondarystructureinotherproteinregionswithouttertiarystructureswerecalculatedby

theintegratedprograms,RVP-netandPSIPRED,respectively.

Annotationofcatalytickinasesofproteinphosphorylationsites

In addition to the experimental annotations of catalytic kinases of protein phosphorylation, we applied

KinasePhos-likepredictionmethod[11-13,17]foridentifying20typesofPTM.Figure2givesanexamplethatthe

experimentalphosphorylationsiteS892of IRS1waspredictedtobecatalyzedbyproteinkinaseMAPKandCDK

withthe preferenceof prolineoccurredon position-2and+1 surrounding thephosphorylation site(position0).

Besides,Y896is predictedtobe catalyzed bykinaseIGF1R, theresultis consistentwithprevious investigation


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[26]. Moreover, S892 is a protein variation site, which was mapped to a non-synonymous single nucleotide

polymorphism(SNP),basedontheannotationobtainedfromdbSNP[27].

EvolutionaryconservationofPTMsites

InordertodeterminewhetheraPTMsitesisconservedamongorthologousproteinsequences,weintegratedthe

databaseofClustersofOrthologousGroups(COGs)[28],whichcollected4873COGsin66unicellulargenomes

and4852clustersofeukaryoticorthologousgroups(KOGs)in7eukaryoticgenomes.ClustalW[29]programwas

adopted to implement the alignment of multiple protein sequences in each cluster, and the aligned profile is

providedintheresource.Anexperimentallyverifiedacetyllysinelocatedinaprotein-conservedregionindicatesan

evolutionaryinfluenceinwhichorthologoussitesinotherspeciescouldbeinvolvedinthesametypeofPTM(See

Additional file 1 - Figure S2). Furthermore, as the example shown in Figure 2, two experimentally verified

phosphorylationsitesareconserved.

PTMbenchmarkdatasetforbioinformaticsstudy

Duetothehigh-throughputofmassspectrometryinproteomics,theexperimentalsubstratesequencesofmorethan

tenPTMtypes,suchasphosphorylation,glycosylation,acetylation,methylation,sulfationandsumoylation,were

investigatedandusedfordevelopingthepredictiontools[14].Tounderstandthepredictiveperformanceof these

toolspreviously developed, it iscrucial to have a common standard for evaluating thepredictive performance

amongvariouspredictiontools.Therefore,weconstructedabenchmark,whichcomprisetheexperimentalsubstrate

sequencesforeachPTMtype.

TheprocesstocompiletheevaluationsetsisdescribedinFigureS3(SeeAdditionalfile1-FigureS3),based

oncriteriadevelopedbyChenetal.[30].Toremovetheredundancy,theproteinsequencescontainingthesame

typeofPTMsitesaregroupedbyathresholdof30%identitybyBLASTCLUST[31].Iftheidentityoftwoprotein

sequencesisgreaterthan30%,were-alignedthefragmentsequencesofthesubstratesbyBL2SEQ.Ifthefragment

sequencesoftwo substrates with the same locationare identical, only one ofthe substratewas includedin the

benchmarkdataset.Therefore,twentyPTMtypescontainingmorethan30experimentalsiteswerecompliedinthe

benchmarkdataset.

Enhancedwebinterface

Auser-friendlywebinterfaceisprovidedforsimplesearching,browsing,anddownloadingofproteinPTMdata.In

additiontothedatabasequerybytheproteinname,genename,UniProtKB/Swiss-ProtIDoraccession,itallows

the input of protein sequences for similarity search against UniProtKB/Swiss-Prot protein sequences (See

Additionalfile1-FigureS4).ToprovideanoverviewofPTMtypesandtheirmodifiedresidues,asummarytable


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isprovidedforbrowsingtheinformationandtheannotationsaboutthepost-translationalmodificationtypes,which

are referred to theUniProtKB/Swiss-Prot PTM list (http://www.expasy.org/cgi-bin/lists?ptmlist.txt) and RESID

[10].

Figure 3 shows an example that users can choose the acetylation of lysine (K) to obtain more detailed

informationsuchasthepositionofmodifiedaminoacid,thelocationof themodificationinproteinsequence,the

modifiedchemicalformula,themassdifference,andthesubstratesitespecificity,whichisthepreferenceofamino

acidssurroundingthemodificationsites.Furthermore,thestructuralinformation,suchassolventaccessibilityand

secondarystructuresurroundingthemodifiedsites,areprovided.AlltheexperimentalPTMsitesandputativePTM

sitescanbedownloadedfromthewebinterface.

Conclusions The proposed server enables both wet-lab biologists and bioinformatics researchers to easily explore the

informationabout protein post-translational modifications. This study not only accumulates the experimentally

verifiedPTMsiteswithrelevantliteraturereferences,butalsocomputationallyannotatestwentytypesofPTMsites

against UniProtKB/Swiss-Prot proteins. As given in Table 2, the proposed knowledge base provides effective

informationof proteinPTMs,including sequenceconservation, subcellular localization andsubstratespecificity,

theaveragesolventaccessibilityandthesecondarystructuresurroundingthemodifiedsite.Moreover,weconstruct

aPTMbenchmarkdatasetthatcanbeadoptedforcomputationalstudiesinevaluatingthepredictiveperformance

of various tools about determining PTM sites. Previous investigations have indicated that many protein

modificationscausebindingdomainsforspecificprotein-proteininteractiontoregulatecellularbehavior[32].All

the experimental PTM sites and putative PTM sites are available and downloadable in the web interface.

ProspectiveworkofdbPTMistointegrateprotein-proteininteractiondata.

Availabilityandrequirements

Projectname:dbPTM2.0:AKnowledgeBaseforProteinPost-TranslationalModifications

ASMDprojecthomepage:http://dbPTM.mbc.nctu.edu.tw/

Operatingsystem(s):Platform-independent

ProgrammingLanguage:PHPPerl

Otherrequirements:amodernwebbrowser(withCSSandJavaScriptsupport)

Restrictionstousebynon-academics:None


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Listofabbreviations

PTM:Post-TranslationalModification;HMMs:hiddenMarkovmodels;PDB:ProteinDataBank;SNP:single

nucleotidepolymorphism.

Competinginterests

Theauthorsdeclarethattheyhavenocompetinginterests.

Authors'contributions

HDHconceptualizedtheproject.TYLandHDHdesignedandbuiltthedatabase.TYL,PCHandWCCperformed

dataanalysis.TYLandJBKHdesignedandbuilttheinterfaces.TYL,JBKHandTYWcompiledapreviousversion

ofthedatabase.HDH,TYLandWCCwrotethedraft.Allauthorstestedthedatabaseandinterfaces.Allauthors

readandapprovedthefinalmanuscript.

Acknowledgements

TheauthorswouldliketothanktheNationalScienceCounciloftheRepublicofChinaforfinanciallysupporting

thisresearchundercontractNo.NSC95-2311-B-009-004-MY3andNSC97-2627-B-009-007.

Special thanks for

financialsupportfromtheNationalResearchProgramforGenomicMedicine(NRPGM),Taiwan.Thisworkwas

alsopartiallysupportedbyMOEATU.Funding topaytheOpenAccesspublicationcharges forthis articlewas

providedbyNationalScienceCounciloftheRepublicofChinaandMOEATU.


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Figures

Figure1-Thesystemarchitectureoftheknowledgebaseforproteintranslational

modification.

Itcomprisesthethreemajorcomponents:integrationofexternalexperimentalPTMdatabases,learningand

predictionof20typesofPTM,andannotationsofPTMknowledge(moredetailsinthetext).

Figure2-Apartofresultpageonthewebinterface.

AnexampleofgraphicalpresentationofPTMsitesandthestructuralcharacteristicsofhumanproteinIRS1.

Figure3-Anillustrativeexampletoshowthecatalyticspecificityofacetyllysine.


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Tables

Table1-ThestatisticsofexperimentalPTMsitesandputativePTMsitesinthisstudy.

PTMtypes Modifiedresidues

No.of

experimentalsites

No.ofputative

sitesfrom

UniProtKB/Swis

s-Prot

No.of

HMM-predictedsitesindbPTM

Phosphorylation Serine,threonine,tyrosine,andhistidine 31,363 36,080 1,815,472

N-linked

GlycosylationAsparagineandlysine 3,264 77,571 179,955

O-linked

Glycosylation

Lysine,praline,serine,threonine,and

tyrosine1,896 2,558 386,545

C-linked

GlycosylationTryptophan 53 52 4,015

AcetylationN-terminalofsomeresiduesandsidechain

oflysineorcysteine 2,080 5,143 1,206

Amidation

GenerallyattheC-terminalofamature

activepeptideafteroxidativecleavageof

lastglycine

2,150 1,117 24,352

Hydroxylation Generallyofasparagine,aspartate,proline

orlysine 1,033 1,074 9,743

Methylation

GenerallyofN-terminalphenylalanine,

sidechainoflysine,arginine,histidine,

asparagineorglutamate,andC-terminal

cysteine

746 2,846 18,716

Pyrrolidone

CarboxylicAcid

N-terminalglutaminewhichhasformedan

internalcycliclactam. 598 584 12,322

Gamma-Carboxygl

utamicAcid Glutamate 371 361 1,924

Farnesylation Cysteine 61 216 5,349

Myristoylation Glycine 108 765 10,998

N-Palmitoylation Cysteine 33 1,279 6,554

S-Palmitoylation Cysteine 177 2,303 21,287

Geranyl-geranylati

onCysteine 47 819 14,317

S-diacylglycerol

cysteineCysteine 36 1,529 8,977

GPIanchoring C-terminalasparagine,asparate,andserine 27 681 -

DeamidationAmidatedasparagineandglutamine(needs

tobefollowedbyaG)38 26 2,022

Sulfation Serine,threonine,andtyrosine 196 626 15,654

Sumoylation Lysine 77 259 10,342

Ubiquitylation Lysine 286 516 8,865

ADP-ribosylation Arginine 3 203 -

Formylation OftheN-terminalmethionine 28 35 -

Citrullination Arginine 27 91 -

Nitration Tyrosine 47 5 1,432Bromination Tryptophan 18 3 -

FAD

O-8alpha-FADtyrosine,Pros-8alpha-FAD

histidine,S-8alpha-FADcysteine,and

Tele-8alpha-FADhistidine

12 116 -

S-nitrosylation Cysteine 9 93 -

Others 1049 2,958 -

Total 45,833 139,909 2,560,047


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Table2-TheenhancedfeaturesinthisexpandingPTMdatabase(dbPTM2.0).

Features PreviousPTMdatabase[11] dbPTM2.0

Proteinentry UniProtKB/Swiss-Prot(release46) UniProtKB/Swiss-Prot(release55)

ExperimentalPTMresource

UniProtKB/Swiss-Prot,Phospho.ELM,andO-GLYCBASE

UniProtKB/Swiss-Prot,Phospho.ELM,

PHOSIDA,HPRD, O-GLYCBASE,and

UbiProt

Computationally

predictedPTMs

Phosphorylation,glycosylation,and

sulfation

About25typesofPTM(phosphorylation,

glycosylation,sulfation,acetylation,

methylation,sumoylation,hydroxylation,

etc.)

Proteinstructure ProteinDataBank(PDB) ProteinDataBank(PDB)

PTMannotation RESID(373PTMannotations) RESID(431PTMannotations)

Structuralinvestigation

ofPTMsites-

Solventaccessibility, secondarystructure

andintrinsicdisorderregion

Kinasefamily

annotation- KinBase

Proteindomain InterPro InterPro

Proteinvariation Swiss-ProtandEnsembl Swiss-ProtandEnsembl

Site-specificPTM

literature-

ExtractingthePTM-relatedliteraturesfrom

UniProtKB/Swiss-Prot,Phospho.ELM,

HPRD, O-GLYCBASE,andUbiProt

Substratespecificity -Aminoacidfrequency,solventaccessibility,

secondarystructureanddisorderregion

surroundingmodifiedsites

Evolutionary

conservationofPTM

sites

- COGandClustalW

PTMbenchmarksetfor

computationalstudies-

Providingthebenchmarkforconstructing

PTMtestsettocomparethepredictive

performanceofpredictiontools

Relationshipbetween

PTMandsubcellular

localization

- Analyzing the relationship between PTMandsubcellularlocalization

Graphicalvisualization

PTM, solvent accessibility, secondary

structure, protein variation, protein

domain,andtertiarystructure

PTM,solventaccessibility,secondary

structure,proteinvariation,proteindomain,

tertiarystructure,orthologousconserved

regions,substratesitespecificityandprotein

interactionnetwork


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Additionalfiles

Additionalfile1

Fileformat:DOC

Title:Supplementaryfigures(S1,S2,S3,andS4)andtables(S1,S2,andS3)

Description: The data provided 4 figures and 3 tables. The description of each figures and

tablesaregivenbelow.FigureS1.ThedetailedprocessingflowofKinasePhos-likemethods.

FigureS2.Themultiplesequencealignmentoforthologousconservedregions.FigureS3.The

flowcharttoremovedataredundance.FigureS4.Exampleofsearchwebpages.TableS1.Data

statisticsoftheintegratedresources.TableS2.Theparametersandpredictiveperformanceof

thetrainedmodelswithbestaccuracyforeachPTMtype.TableS3.Thelistofintegrated

databasesandprograms.


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Additional files provided with this submission:

Additional file 1: dbptm2_bmc rn_additional filet_20090226_wenchi.doc, 1023Khttp://www.biomedcentral.com/imedia/1323127087257761/supp1.doc
http://www.biomedcentral.com/imedia/1323127087257761/supp1.dochttp://www.biomedcentral.com/imedia/1323127087257761/supp1.doc

a comprehensive resource for integrating and displaying protein ptms

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