Benefits of Iterative Searches of Large Databases to Interpret Large Human Gut Metaproteomic Data Sets - Bassignani et al. - Unknown - U

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SUPPORTINGINFORMATIONFORBenefitsofIterativeSearchesofLargeDatabasestoInterpretLargeHumanGutMetaproteomicDataSetsArianeBassignani,†,‡,¶,§SandraPlancade,*,§,MagaliBerland,†MelisandeBlein-Nicolas,¶AlainGuillot,‡DidierChevret,‡ChloéMoritz,‡SylvieHuet,§SalwaRizkalla,k,⊥KarineClément,k,⊥JoëlDoré,‡OlivierLangella,*,¶andCatherineJuste,*,‡†UniversitéParis-Saclay,INRAE,MGP,78350,Jouy-en-Josas,France;‡UniversitéParis-Saclay,INRAE,AgroParisTech,MicalisInstitute,78350,Jouy-en-Josas,France.;¶UniversitéParis-Saclay,INRAE,CNRS,AgroParisTech,GQE–LeMoulon,91190,Gif-sur-Yvette,France;§MaIAGE,INRAE,UniversitéParis-Saclay,78350Jouy-en-Josas,France;INRAE,UR875MIAT,F-31326Castanet-Tolosan,France;kSorbonneUniversité,Inserm,UMRSNutritionetObésités;approchessystémiques,Paris,France;⊥AssistancePubliqueHôpitauxdeParis,ServicedeNutrition,CRNHIle-de-France,Pitié-SalpêtrièreHospital,Paris,France.*Correspondingauthors*E-mail:sandra.plancade@inrae.fr*E-mail:olivier.langella@universite-paris-saclay.fr*E-mail:catherine.juste@inrae.frS-1

1TABLEOFCONTENTSupportingExperimentalSectionS1.StoolSampleCollectionandProcessingSupportingExperimentalSectionS2.ProteinDigestionandPeptideDesaltingSupportingExperimentalSectionS3.LC-MS/MSAnalysisTableS1.AnalyticalsequenceofthesamplesTableS2.SummaryofthethreesearchstrategiesTableS3.TaxonomicdiversityallowedbyeachofthethreesearchstrategiesFigureS1.ExperimentalDesignFigureS2.Overviewofthethreeinterrogationmethodsusedinthispaper.FigureS3.ClusteringofProteinsusingtheX!TandemGroupingAlgorithmofX!TandemPipelineFigureS4.PlotofthemassdeltadistributionsforthethreemethodsFigureS5.Speciesdistributionamongphylaintheintersectionofthethreesearches(A),orintheadditionalpoolbroughtbythetwo-step(B)andthethree-stepstrategy(C)FigureS6.iPathprojectionofKOentrieshighlightedwiththethreeapproachesFigureS7.DistributionofmetaproteinsasafunctionofKOentriesembeddedFigureS8.Ifpresent,thefunctionaldiversityofmetaproteinsisrelatedtotheownfunctionaldiversityofitscomponentproteins,nottotheirnumberFigureS9.Percentagesofgenes(A)andmetaproteins(B,recallofFigure4A)assignedtothedifferentphylawithinthe48microbiomesFigureS10.Exampleofreproducibilityofthepositioningofreplicatesrelativetoothersamplesinthecohort,basedonrelativeabundancesofallmetaproteinsFigureS11.Metaproteinsoverlappinginallpairsofreplicateswhensingletonsareomitted.S-2

2SUPPORTINGEXPERIMENTALSECTIONSupportingExperimentalSectionS1:StoolSampleCollectionandProcessingAllsampleswereself-collectedaspreviouslydetailed18.About1gstoolaliquotswerecutfrozenandthemicrobiotawasseparatedfromthefecalmatrixbyflotationinapreformedNycodenzcontinuousgradientaccordingtoavariantofthemethodpreviouslydetailedbyJusteetal.18Here,wejustreducedthesizeofthegradient.Briefly,stoolspecimensweresupplementedupto2.82gwith1XPBS0.03%w/vNa-deoxycholate,thenwith8mlofNycodenz60%,and6.5mlofthissuspensionwereloadedbelowapreformedgradientwhichhasbeenpreparedwith5mlofa23%w/vNycodenzsolutioninanUltra-Clearcentrifugetube(14.5X95mm,BeckmanInstruments,CA,USA).Duringlow-speedultracentrifugationinaswingingSW40Tirotor(Beckman,14,567g,45min,4°C),bacterialcellsmigrateduptotheirbuoyantdensity(d1.110-1.190)whiletheunwantedfecalmatrixsedimented.AfterwashingincoldTrissaline(20mMTris,138mMNaCl,2.7mMKCl,0.03%w/vNa-deoxycholate,pH7.4),theextractedmicrobiotawerefrozeninliquidnitrogenthenkeptat-80°Cin2mLscrewcapSarstedttubes.Forbacteriallysis,1.5mLofcoldsalineTris-EDTAbuffer(50mMTris-HCl,pH7.8containing150mMNaCland1mMEDTA,andextemporallysupplementedwithPMSFatafinalconcentrationof2mMandproteaseinhibitorcocktail(cOmpleteTM,EDTA-freeProteaseInhibitorCocktail,ROCHE)atafinalconcentrationof1.3X),wasdirectlyaddedtoeachfrozenbacterialpellet.Thepelletsweredispersedbyvigorousvortexingandsonicatedoniceusinga3mmdiameterprobeinshortintervalsof10secON/10secOFF,with20%amplitude,andfortwo5minperiodsseparatedbya15minbreakonicewithperiodicvigorousvortexing.Finally,thesuspensionswerecentrifugedat5000gfor30minat4°Ctoremoveunbrokencellsandlargecellulardebris.Thesupernatantwereultracentrifugedinaswingingrotor(SW55Ti,Beckman)at220,000gfor30minat4°Ctoseparatecellenvelopes(pellet)andcytosolicfractions(supernatant).S-3

3SupportingExperimentalSectionS2:ProteinDigestionandPeptideDesaltingCellenvelope-enrichedpelletedintheultracentrifugetubeswereresuspendedinaminimalvolume(40µL)of50mMNH4HCO3containing0.2%ProteaseMAXsurfactant(refPromegaV2072),usingaMicromanpipettefittedwitha50µLcapillarypiston,andthensonicatedandvortexeduntilhomogeneoussuspensionswereobtained.Thesuspensionsweretransferredinacetone-compatible2mLProteinLoBindtubes(refEppendorf0030108132),addedwith60µLofultracentrifugetuberinsingNH4HCO3(50mM,detergent-free),andthenprecipitatedonicewithfourvolumesofpureice-cold(-20°C)acetone.Thesuspensionwaschilleddownto-20°Cwithvigorousvortexingat10minintervalsoverthefirst30min,andthenleftat-20°Covernight.Thenextmorning,suspensionsweretransferredfor10minat-80°Cbeforebeingcentrifugedat16,000g,4°Cfor10min.Supernatantsweredecantedandtheproteinpelletswerewashedtwicewithice-cold(-20°C)acetone80%inMilliQwater.Proteinconcentrationwasdeterminedbeforethesecondacetonewashdecanting,usingthe2D-Quantkit(refGEHealthcare80-6483-5,short-circuitingprecipitant/coprecipitantuseofthekitasinourcaseproteinswereprecipitatedbyacetone).Aliquotsequivalentto50µgproteinwerefrozeninliquidnitrogenthenkeptat-80°C.Reduction,alkylation,andliquiddigestioninthepresenceofTrypsinGold(refPromegaV5280)inatrypsin-to-proteinratioof1:50(w:w),andofProteaseMaxasthesurfactantwereessentiallyasrecommendedinthePromegatechnicalbulletin(https://www.promega.com/-/media/files/resources/protocols/technical-bulletins/101/proteasemax-surfactant-trypsin-enhancer.pdf?la=en).Finally,peptidemixturesweredesaltedonSep-PaktC18PlusShortcartridges(refWatersWAT036810)with35%acetonitrilethen50%inthefinalelutionsteps.ThefrozeneluateswerelyophilizedonaThermoScientificSavantSpeedVacvacuumconcentrator.S-4

4SupportingExperimentalSectionS3:LC-MS/MSAnalysisHPLCwasperformedonanEksigentNanoLC-Ultrasystem(Eksigent,LesUlis,France).Trypsicdigestionproducts(7µg)wereloaded,concentratedanddesaltedonaprecolumncartridge(BIOSPHEREC18,5µmparticlesize;column:100µmi.d.,2cmlength;NanoSeparationsNieuwkoop,TheNetherlands)with0.1%HCOOHat7.5µL.min-1for3min.Theprecolumncartridgewasconnectedtotheseparatingcolumn(AcclaimPepMap100,3µm,100Å,75µmi.d.50cmlength,ThermoFisherScientfic)andthepeptideswereelutedwithanon-lineargradientfrom5to35%ACNin0.1%HCOOHfor180minat300nL.min-1.OnlineanalysisofpeptideswasperformedwithaQ-exactivemassspectrometer(ThermoFisherScientific),usingananoelectrosprayionsource(non-coatedcapillaryprobe,10i.d.;NewObjective,Woburn,MA,USA).PeptideionswereanalysedusingXcalibur2.1withthefollowingdata-dependentacquisitionsteps:(1)fullMSscan(mass-to-chargeratio(m/z)300to1,400,resolution70,000)and(2)MS/MS(normalizedcollisionenergy=30%,resolution17,500).Step2wasrepeatedforthe12majorionsdetectedinstep1.Dynamicexclusionwassetto60sec.S-5

5SupportingTablesTableS1.AnalyticalsequenceofthesamplesTimeofLC.MS.MSrankwithinthegradientLC.MS.MSDateofinjectionSubjectgradientinjectionmxXMLfileinjectionseriesseriesoperatoroperator2014-07-1709:53:29S32A112CFAL2014_07_16_ENV10_01.mzXML2014-07-1717:26:30S07A113CAAL2014_07_16_ENV10_02.mzXML2014-07-1804:48:26S08A116CAAL2014_07_16_Obomics_S15D0_01.mzXML2014-07-1816:10:18S15A119CAAL2014_07_16_Obomics_S21D0_01.mzXML2014-07-1903:32:09S21A1112CAAL2014_07_16_Obomics_S22D0_01.mzXML2014-07-1914:54:06S22A1115CAAL2014_07_16_Obomics_S26D0_01.mzXML2014-07-2002:16:00S26A1118CAAL2014_07_16_Obomics_S35D0_01.mzXML2014-07-2013:37:57S35A1121CCAL2014_07_16_Obomics_S7D0_01.mzXML2014-07-2100:59:51S32A2124CFAL2014_07_16_Obomics_S8D0_01.mzXML2014-07-2320:17:15S32A3125CFAL2014_07_23_ENV10_03.mzXML2014-07-2410:10:48S38A1128CCAL2014_07_23_ENV10_04.mzXML2014-07-2421:32:46S48A1131CAAL2014_07_23_Obomics_S38D0_01.mzXML2014-07-2508:54:41S49A1134CAAL2014_07_23_Obomics_S48D0_01.mzXML2014-07-2607:38:37S32A4139CFAL2014_07_23_Obomics_S49D0_01.mzXML2014-09-3011:32:37S03A121CCAL2014_09_29_ENV10_05.mzXML2014-09-3022:54:40S04A124CCAL2014_09_30_Obomics_S03D0_01.mzXML2014-09-3000:34:10S32A525CFAL2014_09_30_Obomics_S04D0_01.mzXML2014-10-0110:16:39S11A128CAAL2014_09_30_Obomics_S11D0_02.mzXML2014-10-0614:13:00S32A6211CFAL2014_10_06_ENV10_06.mzXML2014-10-0622:10:18S14A1212CAAL2014_10_06_Obomics_S14D0_01.mzXML2014-10-0716:24:33S25A1215CAAL2014_10_07_Obomics_S25D0_01.mzXML2014-10-0803:46:32S30A1218CAAL2014_10_07_Obomics_S30D0_01.mzXML2014-10-0815:08:27S31A1221CAAL2014_10_07_Obomics_S31D0_01.mzXML2014-10-0902:30:26S33A1224CCAL2014_10_07_Obomics_S33D0_01.mzXML2014-10-0913:52:28S36A1227CCAL2014_10_07_Obomics_S36D0_01.mzXML2014-10-1001:14:20S37A1230CCAL2014_10_07_Obomics_S37D0_01.mzXML2014-10-1313:06:37S32A7233CFAL2014_10_13_ENV10_07.mzXML2014-10-1317:37:37S39A1234CCAL2014_10_13_ENV10_08.mzXML2014-10-1404:59:38S40A1237CCAL2014_10_13_ENV10_08_141018202350.mzXML2014-10-1503:43:54S42A1243CCAL2014_10_13_Obomics_S39D0_01.mzXML2014-10-1515:06:04S43A1246CAAL2014_10_13_Obomics_S40D0_01.mzXML2014-10-1602:28:20S46A1249CAAL2014_10_13_Obomics_S42D0_01.mzXML2014-10-1618:19:31S32A9252CFAL2014_10_13_Obomics_S43D0_01.mzXML2014-10-1622:50:35S47A1253CAAL2014_10_13_Obomics_S46D0_01.mzXML2014-10-1710:13:31S51A1256CCAL2014_10_13_Obomics_S47D0_01.mzXML2014-10-1721:36:54S52A1259CCAL2014_10_13_Obomics_S51D0_01.mzXML2014-10-1808:59:47S53A1261CCAL2014_10_13_Obomics_S52D0_01.mzXML2014-10-1820:23:50S32A8264CFAL2014_10_13_Obomics_S53D0_01.mzXML2015-11-1323:10:02S32B231CADI20151112_S1_D0.mzXML2015-11-1402:35:38S32C132CCDI20151112_S10_D0_A.mzXML2015-11-1500:14:16S01A138CCDI20151112_S13_D0_A.mzXML2015-11-1511:36:21S02A1311CCDI20151112_S16_D0_.mzXML2015-11-1616:28:21S32B1314CADI20151112_S17_D0reinj2.mzXML2015-11-1620:59:21S06A1315CADI20151112_S19_D0_reinj.mzXML2015-11-1723:08:44S09A1320CCDI20151112_S2_D0.mzXML2015-11-1810:30:46S10A1323CADI20151112_S20_D0reinj.mzXML2015-11-1901:18:24S13A1327CCDI20151112_S23_D0_Areinj.mzXML2015-11-1916:05:57S32B3331CADI20151112_S27_D0reinj.mzXML2015-11-1920:36:59S16A1332CADI20151112_S28_D0reinj.mzXML2015-11-2614:18:18S32C2338CCDI20151112_S29_D0reinj2.mzXML2015-11-2713:02:27S19A1344CADI20151112_S32_DO_A_151116162821.mzXML2015-11-2800:24:35S20A1347CADI20151112_S32_DO_A3.mzXML2015-11-2811:46:39S23A1350CADI20151112_S32_DO_A.mzXML2015-11-2902:34:15S27A1354CADI20151112_S32_DO_B2_reinj2.mzXML2015-11-2921:52:49S17A3358CADI20151112_S32_DO_B.mzXML2015-11-3002:23:48S28A1359CADI20151112_S34_D0.mzXML2015-11-3013:45:53S29A1362CADI20151112_S5_D0_reinj.mzXML2015-12-0101:07:52S34A1365CCDI20151112_S6_D0.mzXML2015-12-0112:29:49S44A1368CADI20151112_S9_D0.mzXML2015-12-0206:42:59S45A1373CADI2015112_S44_D0A.mzXML2015-12-0302:01:45S05A1378CCDI2015112_S45_D0A.mzXMLS-6

6TableS2.SummaryofthethreesearchstrategiesTableS3.TaxonomicdiversityallowedbyeachofthethreesearchstrategiesS-7

7SupportingFiguresABFigureS1.ExperimentalDesign.(A)47stoolsampleswereeachpreparedandinjectedonlyonce.(B)Onestoolsamplewaspreparedintriplicates(A,BandC),whichwereinjectednine,threeandtwotimes,respectively.S-8

8ABCFigureS2.Overviewofthethreeinterrogationmethodsusedinthispaper.(A)Classicalone-stepinterrogation.(B)Two-stepiterativeinterrogation.(C)Three-stepiterativeinterrogation.S-9

9ABCFigureS3.GroupingofproteinsusingtheX!TandemGroupingAlgorithmofX!TandemPipeline.(A)Peptides(coloredbands1-12)andproteins(A-F)identifiedbyLC-MS/MS.(B)Theclusteringistogenerateaparsimoniouslistofpeptidesandproteinsidentified.Here,CandFareremovedbecausetheirpresenceisnotconfirmedbythepresenceofspecificpeptides.AandBdefinetwodifferentsubgroupsbecausetheyeachhaveatleastonespecificpeptide(pep4andpep6forA,pep5andpep7forB).AandBalsosharethreepeptides(pep1-3),sothattheyareclusteredwithinthesamegroup.DandEsharealltheirpeptidessothattheyareclusteredwithinthesamesubgroup,buttheysharenopeptidewithAandB,sothattheyareclusteredinadifferentgroup.Onlysubgroupentitieswereusedinthepresentpaper,andreferredasto‘metaproteins’.(C)ExtractoftheoutputofX!TandemPipelineafterclusteringofproteins.Spectralcountsforeachmetaprotein(inrows)andeachsampleincludedintheexperiment(incolumns)aregivenfromcolumn7.S-10

10FigureS4.Distributionofthemassdeltavalues(foreachPSM,thepeptide'scalculatedmassminusitsmeasuredmassinppm)forthewholedataset(48samples),andforeachmethodused.IdentificationswiththethreemethodsfollowthesameGaussiandistribution,whichmeansthatfalsepositiveswerejustaswellcontrolledintheiterativeandtheclassicalmethods.S-11

11200additionalspeciescommoncoreof228additionalspeciespredictedwiththe3-2111speciespredictedwiththe2-stepsearchcomparedtophylapedictedbyallphylaphylastepsearchcomparedthecombinationofthethreesearchtotheclassicalsearchclassicalandthe2-stepstrategiessearchFirmicutes1279Firmicutes119Firmicutes81Bacteroidetes455Bacteroidetes48Bacteroidetes69Proteobacteria147Proteobacteria25Proteobacteria20Actinobacteria113Actinobacteria14Actinobacteria8unclassifiedBacteria21Verrucomicrobia5unclassifiedBacteria6Verrucomicrobia14Spirochaetes4Verrucomicrobia3Fusobacteria11unclassifiedBacteria4Elusimicrobia2Euryarchaeota9Fusobacteria2Lentisphaerae2unclassifiedEukaryota8Basidiomycota1Spirochaetes2Tenericutes7Chloroflexi1Acidobacteria1Spirochaetes6Cyanobacteria1CandidatusOmnitrophica1Chloroflexi5Euryarchaeota1Chloroflexi1Fibrobacteres5Lentisphaerae1Fusobacteria1Lentisphaerae5Nitrospirae1Nitrospinae1Synergistetes4Tenericutes1Synergistetes1unclassifiedViruses4Total228Tenericutes1Acidobacteria2Total200Ascomycota2Chlamydiae2Distributionofthe228additionalspeciesDistributionofthe200additionalspeciesCyanobacteria2amongphylaamongphylaMucoromycota2BFirmicutesCFirmicutesBasidiomycota1BacteroidetesBacteroidetesCandidatusMelainabacteria1CandidatusShapirobacteria1ProteobacteriaProteobacteriaElusimicrobia1ActinobacteriaActinobacteriaIgnavibacteriae12%2%2%1%0%Verrucomicrobia2%1%1%1%1%unclassifiedBacteria3%Neocallimastigomycota16%4%VerrucomicrobiaSpirochaetesThermodesulfobacteria1Elusimicrobia11%unclassifiedBacteria10%41%Zoopagomycota1LentisphaeraeTotal2111Fusobacteria52%SpirochaetesBasidiomycotaFirmicutesAcidobacteriaDistributionofte2111speciesBacteroidetesChloroflexi21%CandidatusOmnitrophicaProteobacteriaCyanobacteriaamongphylaActinobacteria35%ChloroflexiunclassifiedBacteriaEuryarchaeotaFusobacteriaAVerrucomicrobiaLentisphaeraeFusobacteriaNitrospinaeEuryarchaeotaNitrospiraeSynergistetesunclassifiedEukaryotaTenericutesTenericutesTenericutesSpirochaetes1%1%0%Chloroflexi5%Fibrobacteres7%LentisphaeraeSynergistetesunclassifiedVirusesAcidobacteriaAscomycota22%Chlamydiae61%CyanobacteriaMucoromycotaBasidiomycotaCandidatusMelainabacteriaCandidatusShapirobacteriaElusimicrobiaIgnavibacteriaeNeocallimastigomycotaThermodesulfobacteriaZoopagomycotaFigureS5.Speciesdistributionamongphylaintheintersectionofthethreesearches(A),orintheadditionalpoolbroughtbythetwo-step(B)andthethree-stepstrategy(C).S-12

12FigureS6.iPathprojectionofKOentrieshighlightedwiththethreeapproaches:(—)intersectionofallthreeapproaches;(—)intersectionofsupplementalKOentrieswiththetwo-stepandthethree-stepiterativestrategies;(—)supplementalKOentriesspecifictothethree-stepiterativeapproach.Otherminorspecificities(redandblueforKOentriesspecifictotheclassicalandthetwo-stepiterativeapproach,respectively,andvioletforintersectionofboth)canbevisualizedbyzooming.S-13

13ClassicalsearchTwo-stepsearchThree-stepsearchNumberofKOentriespermetaproteinFigureS7.DistributionofmetaproteinsasafunctionofKOentriesembedded:mostmetaproteinshaveonlyoneKOentry.ClassicalsearchTwo-stepsearchThree-stepsearchmetaproteinmetaproteinmetaproteinKOentriesperKOentriesperKOentriesperumberofumberofumberofNNNNumberofproteinmemberspermetaproteinFigureS8.Ifpresent,thefunctionaldiversityofmetaproteinsisrelatedtotheownfunctionaldiversityofitscomponentproteins,nottotheirnumber.S-14

14FigureS9.Percentagesofgenes(A)andmetaproteins(B,recallofFigure4A)assignedtothedifferentphylawithinthe48microbiomes.S-15

15FigureS10.Exampleofreproducibilityofthepositioningofreplicatesrelativetoothersamplesinthecohort,basedonrelativeabundancesofallmetaproteins.Heretwoclusteringsfromthethree-stepiterativesearchstrategywererandomlydrawnamongthefourteen.Thetworeplicates(borderedwithredsolidline)clusterizedwiththesamefivenon-replicatedsamples(borderedwithreddashedline).Thesamefigurewasobservedforallreplicates,andforallthreesearchstrategies.S-16

16FigureS11.Metaproteinsoverlappinginallpairsofreplicates,witheachinterrogationstrategy.Singletons,whicharemetaproteinswithonlyonespecificpeptideofverylowabundance(oneSC)inoneofthetworeplicatesonly,wereomitted.S-17