nature methods doi:10.1038/nmeth...jesse g. meyer1, sushanth mukkamalla1, hanno steen2, alexey i....

Nature Methods doi:10.1038/nmeth.4334

Supplementary Figure 1

Details of PIQED automated qualitative and quantitative post-translational modification analysis workflow.

Modified peptides enriched from biological samples or peptides from proteome digestion without modification enrichment are analyzed by data-independent acquisition. PIQED supports data from SCIEX or Thermo instruments. PIQED automates all data analysis steps st arting from instrument .wiff or .raw files, including: (1) file conversion and pseudo-MS/MS spectra generation using DIA-Umpire, (2) database searching by MS-GF+, X! Tandem, and/or Comet followed by results refinement and combination using PeptideProphet/iProphet/PTMProphet, (3) automated spectral library generation and fragment area extraction using SkylineRunner, and finally, (4) Skyline report generation, filtering for only localized PTMs with certain iProphet and PTMProphet scores, option protein level correction, and table reformatting for significance testing with mapDIA. All files produced along the analysis are output by the pipeline, including the raw search outputs, the various refined pep.xml files, the Skyline file containing extracted peaks for manual review, and the final output from mapDIA, which includes site-level fold change for all desired comparisons.



Screenshot of the open-source PIQED GUI written in Java, Windows Batch, Python, and R.

The software has several optional modules that can be run, where each subsequent module can automatically use the output from the previous module. These modules are broadly divided into three sections: (1) file conversions and DIA-Umpire signal extraction, (2) database searches, and (3) post processing, quantification and significance testing. The leftmost section is used to specify parameters for file conversions and DIA-Umpire signal extraction module that generates pseudo-MS/MS spectra for database searches. The middle section is used to set the database search parameters for MS-GF+, X!Tandem, and Comet. The right section contains input for PeptideProphet, iProphet, PTMProphet, Skyline peak area report generation, and mapDIA significance testing. The program user can save and load up to four default parameter sets.



Example of quantitative difference discovered using PIQED for the discovery of phosphorylation sites and changes using previously published Q-Exactive DIA data collected from urine samples without phospho-enrichment.

(A) Annotated spectra for the peptide TCVADEpSAENCDK, which contains phosphoserine pSer-82 from human serum albumin (HSA). (B) Example of extracted ion chromatograms from the precursor (MS1) and fragment (MS2) ions showing excellent coelution and high mass accuracy. (C) Plot of total MS1 and MS2 peak areas for all replicates measured among the three diagnosis groups: diagnosed urinary tract infections (UTI, n=11), diagnosed ovarian cyst (OC, n=11), or control (pain not diagnosed as UTI or OC, n=11). Group average of (D) raw site-level signal, (E) HSA protein-level, and (F) site-level corrected by protein level and local TIC. Error bars are standard error. Although the protein level of HSA is not significantly different between the diagnosis groups, we observed a statistically significant increase in abundance of pSer-82 comparing the UTI group with the undiagnosed pain group with and without the normalization options available in PIQED.



Comparison of quantitative results for HSA and HSA pSer-82 using various normalization options in PIQED.

(A) Plot showing individual total site-level peak areas for the peptide TCVADEpSAENCDK containing phosphoserine pSer-82. (B) Protein-level areas computed with mapDIA using unmodified HSA peptides. (C) Site-level areas (from A) for the peptide TCVADEpSAENCDK containing phosphoserine pSer-82 corrected by the observed protein-level areas from (B). (D) Site-level areas for phosphoserine pSer-82 from the peptide in (A) corrected by local total ion chromatogram signal (TIC). (E) Site-level areas for phosphoserine pSer-82 from the peptide in (A) corrected by local total ion chromatogram signal (TIC) and by the protein-level area from (B). In all cases with or without normalizations, this phosphorylation site is determined to be statistically increased in urine from children diagnosed with UTI compared to the undiagnosed pain group.



Examples of high-quality annotated pseudo-MS/MS spectra showing 4/34 phosphorylation sites in osteopontin identified using PIQED. Osteopontin is overexpressed in a variety of cancers.

(A) Annotated spectra for pSer-219 showing the presence of phosphate neutral loss on all y-ions and a prominent fragment ion corresponding to fragmentation n-terminal of proline. (B) Annotated spectra for a peptide containing pSer-310 from the Osteopontin C-terminal, which has a nearly-complete b-ion series. (C) Annotated spectra for a peptide containing pSer-308 and pSer-310 from the Osteopontin c-terminal, which also contains a nearly complete b-ion series and neutral losses of one or two phosphoric acids starting at b7 and b9, respectively. The spectra in (B) and (C) also contain unlabeled fragment ions that correspond to a neutral loss of water from one of the unmodified serine residues.


SupplementalInformationfor

AutomatedIdentification&QuantificationofProteinModificationsfromData-IndependentAcquisitionJesseG.Meyer1,SushanthMukkamalla1,HannoSteen2,AlexeyI.Nesvizhskii3,4,

BradfordW.Gibson1,BirgitSchilling1*

Affiliations

1BuckInstituteforResearchonAging,Novato,CA,USA.

2DepartmentofPathology,BostonChildren'sHospitalandHarvardMedicalSchool,Boston,Massachusetts,USA.

3DepartmentofComputationalMedicineandBioinformatics,UniversityofMichigan,AnnArbor,Michigan,USA.

4DepartmentofPathology,UniversityofMichigan,AnnArbor,Michigan,USA.

*Correspondingauthor

BirgitSchilling,[email protected]


SUPPLEMENTALNOTE

DETAILEDDESCRIPTIONOFPIQEDANALYSISWORKFLOW

FilesfromDIAareprocessedusingourcustom,open-sourcesoftwarePIQED,whichprovidesagraphical

userinterfaceactingasawrapperforallthecomputationalstepsofcombineddiscoveryand

quantificationofPTMpeptidesfromDIA.TheGitHubrepositorycontainsdetailedinstallationandusage

instructions(https://github.com/jgmeyerucsd/PIQEDia/blob/master/Tutorial_and_manual.pdf).Data

fromeitherSCIEXorThermoFisherScientificinstrumentsarecurrentlysupportedbyPIQED.Raw

instrumentfilesarefirstconvertedto.mzXMLusingmsconvert.exefromProteoWizard,andtheDIA-

Umpiresignalextractionmoduleisusedtogeneratepseudo-MS/MSspectra.MGFfilesproducedfrom

DIA-Umpireareautomaticallyconvertedto.mzXML,andtheusercanchoosetospecifyparametersfor

databasesearchesusinganycombinationofMSGF+1,Comet2,and/orX!Tandem3(seesupplemental

files“comet.Kac.DIA.params”and“xTandem_Kac_params.xml”).ThedatabasesearchresultsfromX!

TandemandMSGF+areautomaticallyconvertedtopep.xml,whereasthecometsearchoutputshould

bespecifiedas.pep.xmlintheCometconfigurationfile.Allsearchoutputsareprocessedthrough

PeptideProphet4usingtheTPPcommandxinteract.exeseparately,combinedusingiProphet5,and

refinedforPTMlocalizationusingPTMProphet6.Peptideidentificationsareimportedintoapre-setup

Skyline7documenttemplateusingSkylineRunner.exetogenerateaspectrallibrary,performfragment

ion-levelsignalextraction,andoutputacustomfragmention-levelpeakareareport.TheSkylinereport

isreadintoRandfilteredforonlypeptidescontainingthemodificationofinterestthathaveauser-

definedPTMProphetlocalizationscore,optionallycorrectedforchangesinproteinlevelifthedirectory

containsthefile“proteinlevels.txt”,andreformattedformapDIA.Theoptionalprotein-levelcorrection

usesprotein-levelintensitymeasurementsfromthesamesamplecomputedusingunmodifiedpeptides

(eitherfromaseparateacquisition,orinthecaseoftheUrinedata,fromunmodifiedpeptidesidentified


inthesameacquisition)anddividesallfragment-levelareasforeachmodificationsitebytheirprotein-

levelintensities.Optionalprotein-levelcorrectionwillautomaticallybeattemptedbythesoftwareifthe

directorycontainsthefile“proteinlevels.txt”,anexampleofwhichisavailableonGitHubunderthe

inputsfolder.Onlymodificationsitesfromproteinsfoundintheseparateprotein-levelquantification

areincludedintheoutput,whichiswhythenumberofphosphorylationsitesquantifiedfromtheurine

datasetdecreasedafterprotein-levelcorrection.SeefigureS4A-Cforanexampleofprotein-level

normalization.TheRstepcanalsorenamereplicatestocontaingroupnames,whichautomatically

happensifthefile“namemapping.txt”ispresentintheoutputdirectory(seeGitHubforanexampleof

“namemapping.txt”).Finally,PIQEDrunsmapDIA8toperforminterferencefilteringandtoassess

statisticalsignificanceofthedesiredcomparisons.Thisstepallowstheoptionfortotalion

chromatogramnormalization,orlocal,retention-timebasednormalization(seefigureS4D-Eand

mapDIApublicationfordetails).Insummary,oursoftwaretoolPIQEDprovidesahigh-throughput

automateddataprocessingpipelinethatnotonlycanbeusedtoidentify,butalsoquantify

posttranslationalmodifications,combiningmanyindividualdataprocessingtoolsinanautomated

fashion,whichcanbeexecutedthroughasimpleGUIandinterface.Atthesametime,byusingaDIA-

onlydataacquisitionworkflow,thePTManalysiscanbeperformedusingsmallamountsofbiological

material(asnoextraDDAacquisitionsareneededforlibrarybuilding),andmassspectrometric

instrumenttimeisreducedsignificantly.TheimplementationofproteinlevelnormalizationforPTM

quantificationallowsformorepreciseresults.

DESCRIPTIONOFFILESONGITHUB:INPUTSANDPARAMS

Diaumpire_se_orbi_strict.txt–parametersfileusedforDIA-Umpiresignalextractionfromurinedataset

Diaumpire_se.params–parametersfileusedforDIA-Umpiresignalextractionmoduleusedfortheacetyldatasetincludingthevariablewindowdefinition


20150810.mouse.cc.iRT.fasta–databasefileusedforMS-GF+databasesearchesoftheacetyldatasetandforpopulatingSkylinedocument

20161213.human.fasta–databasefileusedforMS-GF+databasesearchesoftheurinedatasetandforpopulatingtheSkylinedocument

20150810.mouse.cc.iRT_DECOY.fasta–databasefileusedforCOMETandX!Tandemdatabasesearchesoftheacetyldataset

20161213.human_DECOY.fasta–databasefileusedfortheCOMETandX!Tandemdatabasesearchesoftheurinedataset

Comet.Kac.DIA.params–Cometdatabasesearchparametersfileusedforacetyldataset

Comet64.params.orbi.new–Cometdatabasesearchparametersfileusedforurinedataset

taxonomy.xml–FilerequiredforX!Tandemdatabasesearchesspecifyingthelocationofthedatabasefileusedfortheacetyldataset

human_taxonomy.xml–FilerequiredforX!Tandemdatabasesearchesspecifyingthelocationofthedatabasefileusedfortheurinedataset

xTandem_Kac_params.xml–X!Tandemdatabasesearchparametersfileusedfortheacetyldataset

xTandem_pSTY_orbi_params.xml–X!Tandemdatabasesearchparametersfileusedfortheurinedataset

Skyline\default_empty.sky–‘empty’Skylinedocumentcontainingallappropriatesettingsforthetutorialdataset.Foruserdata,thetemplatedocumentshouldbeeditedtoreflecttheinstrumentparametersusedtocollectyourdata.

Skyline\Orbi_empty.sky–‘empty’Skylinedocumentcontainingexamplesettingsfordataextractionfromorbitrapdata.

DESCRIPTIONOFFILESONGITHUB,ACETYLLYSINEDATASETRESULTSFILES:OUTPUTS\ACETYL_MOUSE_LIVER\

fullDIA.final.interact.ptm.pep.xls.xlsx–iProphet-filteredpeptideidentificationresults

fullDIA.final.interact.ptm.pep.xml–finalpeptideidentificationresultsinpep.xmlformat

fullDIA.pt99.mProph.features.csv–completelistofmProphetfeaturescoresproducedwithinSkyline

2016_0826_mapDIA.skyr–customSkylinereportfile

2016_0826_mapDIA.csv–SkylinereportbeforemapDIAfilteringforinterferencesandreformatting

mapDIA_Input.txt–FilteredandreformattedSkylinereportusedforinputtomapDIA

site_level_areas.txt–mapDIAsite-levelareareportcontainingareasusedforcalculationofCVvaluesinfigure1c.


mapDIA_analysis_output.txt–rawmapDIAoutputresultswithsite-levelfoldchangesandprobabilitiesusedtogeneratefigure1d.

DESCRIPTIONOFFILESONGITHUB,URINEPHOSPHORYLATIONDATASETRESULTSFILES:OUTPUTS\URINE\

ptmProphet-output-file.ptm.pep.xml.zip–compressedPTMprophetoutput

iPro-output-file.pep.xml–combinediProphetresultsfromusedforinputtoPTMProphet

noCor_analysis_output.txt–site-levelmapDIAoutputusingnonormalizationusedtoproducesupplementalfigure3A.

proteinlevels.txt–protein-levelquantitiesusedforprotein-levelcorrectionofsite-levelchangesusedtoproducesupplementalfigure3B.

protlvlCor_noTICcor_analysis_output.txt-site-levelmapDIAoutputusingprotein-levelnormalizationbutnotlocalTICnormalizationusedtoproducesupplementalfigure3C.

TICcor_noProtCor_analysis_output.txt-site-levelmapDIAoutputusinglocalTICnormalizationbutnotprotein-levelnormalizationusedtoproducesupplementalfigure3D.

bothCor_analysis_output.txt–site-levelmapDIAoutputusingbothlocalTICnormalizationandprotein-levelcorrectionusedtoproducesupplementalfigure3E.


SUPPLEMENTALMETHODS

SAMPLEPREPARATION–ENRICHMENTOFACETYLATEDPEPTIDESFROMMOUSELIVER

AnimalstudieswereperformedaccordingtoprotocolsapprovedbyIACUC(theInstitutionalAnimalCare

andUseCommittee).Sirt5-/-malemiceon129backgroundweremaintainedonastandardchowdiet

(5053PicoLabdiet,Purina)untiltheyweresacrificedat24weeksofageforexperiments.Livertissue

fromaSirt5knockoutmousewaslysedand20mgofproteinlysatewasdigestedasdescribed

previously9.Acetylatedpeptideswereenrichedusing1tubeofanti-acetyllysineantibody-bead

conjugatedPTMScan(CellSignalingTechnologies)accordingtothemanufacturer’sinstructions.

PeptideselutedfromtheenrichmentweredesaltedusingC18reversedphaseStageTips,and

resuspendedin0.2%formicacidformassspectrometryanalysis.

NANO-LIQUIDCHROMATOGRAPHY-TANDEMMASSSPECTROMETRY

PeptideseparationswerecarriedoutusingmobilephaseAconsistingof97.95%water/0.05%FA/2%

acetonitrile,andmobilephaseBconsistingof98%acetonitrile/1.95%water/0.05%FA.Sampleswere

loadedontoaC18pre-columnchip(200µmx6mmChromXPC18-CLchip,3µm,300Å)usingan

EksigentcHiPLCsystemfor10minutesataflowof2µLperminofmobilephaseA.Separationwas

performedwitha75µmx15cmChromXPC18-CLanalyticalchip(3µm,300Å)usingagradientfrom

95%to60%mobilephaseAover80minutes.Thecolumnchipwaswashedbyanincreaseto80%B

over5minutesthatwasmaintainedfor8minutes,followedbyareturnto95%mobilephaseAovertwo

minutesthatwasmaintainedfor35minutestore-equilibratethecolumn.Elutingpeptideswere

directlyelectrosprayedintoanorthogonalquadrupoletime-of-flightTripleTOF5600massspectrometer

(SCIEX)andanalyzedbydata-independentacquisition(SWATH).EverySWATHcycleconsistedofa250


msprecursorscanfrom400-1,250m/zfollowedbyfragmentationofallionsbetween400-1,200m/z

using64variablewidthprecursorisolationwindows(windowdefinitionscontainedinDIA-Umpiresignal

extractionparametersfile“diaumpire_se.params”)for42mseach,resultinginatotalcycletimeof

approximately3sec.Fragmentionspectrawerecollectedfrom100-2,000m/z.

PIQEDSETTINGSUSEDFOR1XVERSUS2XINJECTIONEXPERIMENT

AllparameterfilesusedinthisstudyareavailablefordownloadfromMassIVE(MassIVEID:

MSV000080189ftp://massive.ucsd.edu/MSV000080189/raw/).TheSkylinefileoutputfromPIQEDhas

beenuploadedtoPanoramaathttps://panoramaweb.org/labkey/project/Schilling/PIQED/begin.view

(useyourgeneralPanaramalogintofreelyaccessthedataset).Pseudo-MS/MSspectrafromDIA-

UmpireweredatabasesearchedusingMSGF+,Comet,andX!TandemagainsttheUniProtdatabaseof

allmouseproteinsdownloadedonAugust10th,2015andreversedsequencestoallowfalsediscovery

rate(FDR)estimationusingthetarget-decoyapproach.SeethesupplementalfilesforCometandX!

Tandemsearchparameters.SearchparametersforMSGF+were:numberoftryptictermini=2,peptide

lengthsfrom7-40aminoacids,chargevaluesfrom1to6,20ppmprecursormasserror,andnoisotope

error.Searchesincludedfixedcarbamidomethylationofcysteineandupto3variablemodificationsby

methionineoxidation,lysineacetylation,peptideN-terminalpyroglutamateformation,andproteinN-

terminalacetylation.Databasesearchresultswereconvertedfrom.mzidto.pep.xmlusing

idconvert.exe,andresultswererefinedusingPeptideProphet.PeptideProphetprocessingusedthe

followingoptions:donotmergefilesintooneanalysis,usePPMmasserrormodel,usethenon-

parametricmodelwithdecoyhitstopindownthenegativedistribution,reportdecoyhitswith

computedprobability,enzymetrypsin,andclevel=0.AfterseparaterefinementwithPeptideProphet,all

databasesearchresultswerecombinedintoonefileusingiProphetwiththedefaultparameters.The


iProphetoutputwasthenusedasinputtoPTMProphetforPTMlocalizationscoring.Finally,thepeptide

identificationresultsin.pep.xmlformatwereimportedintoSkylinetogenerateaspectrallibrary.

SWATHfragmentionpeakareasfromthethreereplicatesof0.5Xand1Xinjectionswereexportedas

thecustomSkylinereportsandpeptidescontaininglysine+42(acetylation)werefilteredbasedona

minimumlocalizationscoreof0.95.mapDIA,whichfiltersfragmentionsanduseswell-established

Bayesianmodelingtocomputethefalsediscoveryratesofchanges,wasthenusedtocomparethetwo

groups(i.e.“halfDIA”and“fullDIA”)withthedefaultparametersusingnonormalization.ThemapDIA

modelisquiterobustwithanydistributionofinputdata,thereforenoassumptionsarerequiredforits

usetotestdifferentialexpression.Theresultspresentedherearerepresentativeofseveralsimilar

experimentsperformedinourlaboratory.

PIQEDANALYSISOFURINEDATAFROMQ-EXACTIVE

Eleveninstrument.rawfilesperdiagnosisgroupweredownloadedfromthesetoffilesavailableon

PeptideAtlas(datasetidentifier:PASS00706)publishedbyMunteletal.10PIQEDsettingforthisdataset

wereasdescribedaboveexceptifnoteddifferentlyhere.Becausethisdatawascollectedwithout

phosphopeptideenrichment,weusedmorestringentsignalextractionandpost-quantificationfiltering

parametersthanthosedescribedintheprevioussection.ThesuggestedDIA-Umpiresignalextraction

parametersincludedwiththeDIA-UmpiredownloadwereusedexceptthatCorrThresholdwassetto

0.5,DeltaApexwassetto0.1,RTOverlapwassetto0.1,BoostComplementaryIonwassettoFALSE,

SE.estimateBGwassettoTRUE,andSE.MassDefectOffsetwassetto0.2.Databasesearcheswere

performedagainstthehumanproteomedownloadedfromUniProtonDecember13th,2016,andused

15ppmprecursortolerance.SearchesallowedvariablephosphorylationofSTYandvariableoxidationof

methionine.


STATISTICS

Acetylatedpeptidedatausedfortheplotsinthemaintextfigurewasfromthreetechnical

replicatescorrespondingtorepeatinjectionsof0.5Xor1Xsamplevolumefromthesame

sample.Theacetylatedpeptideresultspresentedinfigure1arerepresentativeof2repetitions

oftheexperiment.Phosphorylatedpeptidedatacomesfromreanalysisofbiologicalreplicates

correspondingtourinecollectedfromuniquepatients,elevenfromeachdiagnosisgroup(see

supplementalreference10formoresampledetails).Forstatisticaldetailsofeachindividual

softwareprogram,seetheirrespectivepublications.Eachoftheexternalprograms(usedas

partofourpipeline)containstheirownthoroughstatisticalalgorithmsandsignificancetesting.

Noadditionalstatisticaltestswereimplementedoutsideoftheconstitutiveprograms.

CODEAVAILABILITY

ThecurrentPIQEDsoftwareisavailablefromGitHub

(https://github.com/jgmeyerucsd/PIQEDia/releases/tag/v0.1.2),GNUGeneralPublicLicense

v3.0.Updatedandfuturesoftwareversionswillbeavailablefrom

https://github.com/jgmeyerucsd/PIQEDia.

SUPPLEMENTALREFERENCES

1.Kim,S.&Pevzner,P.A.MS-GF+makesprogresstowardsauniversaldatabasesearchtoolfor

proteomics.NatCommun5,(2014).


2.Eng,J.K.,Jahan,T.A.&Hoopmann,M.R.Comet:Anopen-sourceMS/MSsequence

databasesearchtool.PROTEOMICS13,22–24(2013).

3.Craig,R.&Beavis,R.C.TANDEM:matchingproteinswithtandemmassspectra.

Bioinformatics20,1466–1467(2004).

4.Keller,A.,Nesvizhskii,A.I.,Kolker,E.&Aebersold,R.EmpiricalStatisticalModelToEstimate

theAccuracyofPeptideIdentificationsMadebyMS/MSandDatabaseSearch.Anal.Chem.

74,5383–5392(2002).

5. Shteynberg,D.etal.iProphet:Multi-levelIntegrativeAnalysisofShotgunProteomicData

ImprovesPeptideandProteinIdentificationRatesandErrorEstimates.Mol.Cell.Proteomics

10,(2011).

6. Shteynberg,D.etal.PTMProphet:TPPsoftwareforvalidationofmodifiedsitelocationson

post-translationallymodifiedpeptides.in60thASMSConferenceonMassSpectrometry,

Vancouver,BC,Canada20–24(2012).

7. Egertson,J.D.,MacLean,B.,Johnson,R.,Xuan,Y.&MacCoss,M.J.Multiplexedpeptide

analysisusingdata-independentacquisitionandSkyline.NatProtoc.10,887–903(2015).

8. Teo,G.etal.mapDIA:Preprocessingandstatisticalanalysisofquantitativeproteomicsdata

fromdataindependentacquisitionmassspectrometry.J.Proteomics129,108–120(2015).

9.Rardin,M.J.etal.SIRT5regulatesthemitochondriallysinesuccinylomeandmetabolic

networks.CellMetab.18,920–933(2013).

10.Muntel,J.etal.AdvancingUrinaryProteinBiomarkerDiscoverybyData-Independent

AcquisitiononaQuadrupole-OrbitrapMassSpectrometer.J.ProteomeRes.14,4752–4762

(2015).


nature methods doi:10.1038/nmeth...jesse g. meyer1, sushanth mukkamalla1, hanno steen2, alexey i....

Documents