Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human tissue samples

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Aalborg Universitet

Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed

paraffin-embedded human tissue samples

Bennike, Tue Bjerg; Kastaniegaard, Kenneth; Padurariu, Simona ; Gaihede, Michael;

Birkelund, Svend; Andersen, Vibeke; Stensballe, Allan

Published in:

EuPA Open Proteomics

DOI (link to publication from Publisher):

10.1016/j.euprot.2015.10.001

Publication date:

2016

Document Version

Publisher's PDF, also known as Version of record

Link to publication from Aalborg University

Citation for published version (APA):

Bennike, T. B., Kastaniegaard, K., Padurariu, S., Gaihede, M., Birkelund, S., Andersen, V., & Stensballe, A.

(2016). Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded

human tissue samples. EuPA Open Proteomics, 10, 9-18. DOI: 10.1016/j.euprot.2015.10.001

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$

Comparing

the

proteome

of

snap

frozen,

RNAlater

preserved,

and

formalin-

fixed

paraf

fin-embedded

human

tissue

samples

Tue

Bjerg

Bennike

a,b,1

,

Kenneth

Kastaniegaard

b,1

,

Simona

Padurariu

c

,

Michael

Gaihede

c,d

,

Svend

Birkelund

b

,

Vibeke

Andersen

a,e,2

,

Allan

Stensballe

b,

*

,2

a

ResearchUnitforMolecularDiagnosticandClinicalResearch,HospitalofSouthernJutland,Aabenraa,Denmark

b

DepartmentofHealthScienceandTechnology,AalborgUniversity,Aalborg,Denmark

cDepartmentofOtolaryngology,HeadandNeckSurgery,AalborgUniversityHospital,Aalborg,Denmark, dDepartmentofClinicalMedicine,AalborgUniversity,Aalborg,Denmark

e

InstituteofRegionalHealthResearch-CenterSoenderjylland,UniversityofSouthernDenmark,Odense,Denmark

ARTICLE INFO Articlehistory: Received24June2015

Receivedinrevisedform19August2015 Accepted25October2015

Availableonline2November2015

Keywords: Proteomics RNAlater Formalin-fixed Paraffin-embedded Humancolonmucosa Preservation Massspectrometry

ABSTRACT

Largebiobanksexistworldwidecontainingformalin-fixed,paraffin-embeddedsamplesandsamples storedinRNAlater.However,theimpactoftissuepreservationontheresultofaquantativeproteome analysisremainspoorlydescribed.

Humancolonmucosalbiopsieswereextractedfromthesigmoideumandeitherimmediatelyfrozen, stabilizedinRNAlater,orstabilizedbyformalin-fixation.Inonesetofbiopsies,formalinstabilizationwas delayed for 30min. The protein content of the samples was characterized by high throughput quantitativeproteomics.

Wewereabletoidentifyasimilarhighnumberofproteinsinthesamplesregardlessofpreservation method,withonlyminordifferencesinproteinquantitation.

ã2015TheAuthors.PublishedbyElsevierB.V.onbehalfofEuropeanProteomicsAssociation(EuPA).This isanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

1.Introduction

Clinicalproteomicresearchisdependentontheavailabilityof clinicalsamples.Proteomicscanprovideinformationconcerning diseaseetiology,biomarkersfordiagnosis,responsetotherapyand noveldrugdiscovery[1].Arapidstabilizationofthesamplewith eliminationofenzymaticandcellactivityiscriticaltopreservethe biological state of the material. A commonly used method for samplestabilizationisbydirectlyfreezing(DF)thesampleswith liquidnitrogenat 196Cordryiceat 79C,whichpreservesthe sample with minimal introduction of chemical modifications. BiobankscontainingDFsamples,formalin-fixed,paraf fin-embed-ded (FFPE) tissue samples, and samples stored in RNAlater constitutevastsourcesof samples,whereofespeciallythelatter remainslargelyunexploitedforproteomicsanalysis[2,3].Dueto standard hospitaland clinicprotocols a delaybetween sample extraction and sample stabilization can be expected for most samples in biobanks [4]. However, the impact of RNAlater preservationonhumantissuesamplesontheresultofaquantative proteome study, as well as the impact of delaying tissue stabilization,remainspoorlydescribed.

Abbreviations:CAN,acetonitrile;FA,formicacid;FDR,falsediscoveryrate;DF, directly-frozen;FASP,filter-aidedsamplepreparation;FFPE,formalin-fixed;HLA-A class I, histocompatibility antigen A-23 alpha chain; HLA-DRB1 class II, histocompatibility antigen DRB1-4 beta chain; LFQ, label-free quantification; iFFPE, immediately formalin-fixed; PCA, principle component analysis; PSM, peptidespectralmatch;PTM,post-translationalmodification;s,standard devia-tion;sFFPE,storedfor30minpriortoformalin-fixed;SDC,sodiumdeoxycholate; SDS,sodiumdodecylsulfate;TEAB,triethylammoniumbicarbonate.

Significance:Wehavedemonstratedthefeasibilityinconductingproteomeanalysis ofsamplesstabilizedinRNAlaterandformalinfixed,paraffin-embeddedsamples, andproposeanalysisstrategiesforboth.EspeciallyRNAlaterpreservationwas foundtobeapromisingalternativetosnapfreezingsamplesforproteomicsstudies, making a simple and uniform sample preservation possible for proteomic, transcriptomic,andgenomicstudies.Delayingtissuestabilizationwith formalin-fixationfor30minonlyhadaminorimpactontheresultoftheanalysis.Ourstudy demonstratedthefeasibilityinconductinganalysisofsamplesstoredinbiobanksto extrapolateretrospectiveinformationforstudiesindiagnosis,responsetotherapy, andnoveldrugdiscovery.

* Correspondingauthor.DepartmentofHealthScienceandTechnology,Aalborg University,FredrikBajersVej3B,9220Aalborg,Denmark.Fax:+4598154008.

E-mailaddress:as@hst.aau.dk(A.Stensballe).

1

Contributedequallytothemanuscript.

2

Sharedlastauthors.

http://dx.doi.org/10.1016/j.euprot.2015.10.001

2212-9685/ã2015TheAuthors.PublishedbyElsevierB.V.onbehalfofEuropeanProteomicsAssociation(EuPA).ThisisanopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

EuPAOpenProteomics10(2016)9–18

ContentslistsavailableatScienceDirect

EuPA

Open

Proteomics

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RNAlater is primarily used to stabilize the RNA content of samples for clinical genomic and transcriptomic analysis, and samplesstoredinRNAlaterhavebeenusedextensivelyinDNAand RNAstudies.TheRNAlatersolutioncontainshighconcentrationof quaternaryammoniumsulfatesandcesiumsulfatewhich dena-tureproteins,includingDNases,RNases, andproteases, thereby stabilizingtheDNA,RNA,andproteincontent[5,6].Afewstudies have investigated the feasibility in retrieving proteins from samplesstoredinRNAlater[5,7–10].Saitoetal.havedemonstrated the feasibility of retrieving proteins from bacteria stored in RNAlaterwithahighnumberofidentifiedproteins,andasimilar relativeproteinabundancecomparedwithfrozenbacteria[9].Han etal.[10]haveperformeda protein-basedbiomarkerdiscovery studyonhumantissuestoredinRNAlateridentifyinghundredsof different proteins. However, no studies have investigated the impactontheextractedproteinabundancesusinghumantissue samples,northeimpactonthepost-translationalmodifications (PTMs)ofpreservingsamplesinRNAlater,whichiscriticalforthe subsequentdataanalysis.Thefeasibilityinconductingareliable proteome analysis of tissue stored in RNAlater would make coupledtranscriptomics and proteomics analysis accessible for rigorousandcomprehensivemolecularassessment[9].

The stability of FFPE tissue mainly arises due to molecular crosslinkingof proteinswhich are establishedduring formalin-fixation[11,12].Thecrosslinksariseastheproductofathreestep modification:(1)formalinreactswiththeaminoorthiolgroupson theaminoacidsleadingtomethyloladditions.(2)Themethylol adduct on the primary amino groups is partially dehydrated leadingtotheformationoflabileSchiffbases,(3)whichcanform crosslinksbetweenseveralaminoacids,i.e.,arginine,asparagine, cysteine, glutamine, histidine, tryptophan, and tyrosine [11,12]. FFPEsamplesarehighlystableandthehistologicaland morpho-logicalarchitectureofthetissue ispreserved[2,13].Asaresult, samplesareroutinelyacquiredasclinicaldiagnosticbiopsiesand largerepositorieshavebeengeneratedworldwide[2,13].Several proteomicstudieshavedemonstratedthefeasibilityinextracting and identifying proteins from FFPE samples using proteomics techniques[2,3,14–21].

Theaims ofthestudyweretoinvestigate theimpactonthe resultofa quantative proteomestudyof(1) preserving human tissue samples in RNAlater compared to DF and FFPE, and (2) delaying tissue stabilization for 30min, in terms of protein abundances, protein modifications, and protein denaturation. We, therefore, examined methods for protein extraction from tissuepreservedinRNAlatersolutionorbyFFPE,withDFtissueasa control, comparing the result of a LC–MS/MS-based proteome analysis. We focused ouranalysis ona set of softtissue colon biopsiesextractedforthepurposeofthisstudytodemonstratethe potentialsinbiobankanalysiswiththesuggestedprotocols.The filter-aidedsamplepreparation(FASP)methodiscommonlyused for preparing samples prior tobottom-up proteomics analysis. MolecularcutoffspinfiltersarecentralintheFASPprotocol,which facilitatesefficientandeasilyconductedbufferchanges,beneficial inrelationtoproteinextractionfromtissueinRNAlatersolutionto remove salts [22–24]. For FFPE samples we adapted the de-crosslinkingmethodfromWakabayashietal.[16]foruseinaFASP protocol. Additionally, we implemented a buffer optimization recommendedbyKawashimaetal.[25]toenhancetheyieldof proteinextraction.

2.Materialsandmethods 2.1.Collectionofsamplematerial

Colonmucosalbiopsiesweresampledfromthesigmoideumof twogastroenterologicalhealthypersons,byendoscopyatHospital

of Southern Jutland, Aabenraa,Denmark. Twelve biopsieswere extractedfromeachpersonapproximately40cmfromtheanus, keptconstantforeachperson.Allbiopsieshadanapproximatesize of 1–2mm3, and thebiopsieswere preservedby fourdifferent

methods.Directlyfrozenbiopsies(DF)wereimmediately trans-ferredtoindividualcryotubesandwithin10–20ssnapfrozenwith liquidnitrogenfollowedbystorageat 80C.RNAlaterbiopsies were immediately transferred to individual cryotubes prefilled with0.5mLRNAlater(LifeTechnologies,Carlsbad,CA,USA),stored at room temperature for 24h followed by storage at 80C, according to manufacturer’s instructions. FFPE biopsies were following extraction within 10–20s placed in preparation car-tridges.Biopsieswereeitherimmediately(iFFPE)stabilizedin4% formaldehyde,orstoredfor30minatambienttemperaturebefore stabilizationwith4%formaldehyde(sFFPE)tosimulateaclinical situation. Paraffin embedding was performed after a week at DepartmentofPathology,AalborgUniversityHospital,Denmark, accordingtocurrentstandards.Allsampleswerestoredforatotal ofonemonthpriortoproteomicssamplepreparationandanalysis. The projectwas approvedby The Regional Scientific Ethical Committee(S-20120204)andtheDanishDataProtectionAgency (2008-58-035),andallparticipantshadgiveninformedconsentto participateinthestudy.

2.2.Proteomicssamplepreparation

WeutilizedamodifiedFASPtrypticproteindigestionprotocol forthesamplepreparation,withethylacetatephaseinversionto facilitatesurfactantremoval[22–24,26,27].Wakabayashietal.[16]

utilized a lysis buffer with 100mM Tris–HCl for the protein extraction.However,Kawashimaetal.[25]foundthatincreasing the concentration of Tris–HCl in the lysis buffer to 300mM significantly improved the efficiency of the protein extraction, whichweimplementedintheFASPprotocol.

RNAlater and DF preserved samples were homogenized in 0.5mL lysis buffer (12mM sodium deoxycholate (SDC), 12mM sodiumdodecylsulfate (SDS)in 300mM Tris/HCl,pH9.0)with steelbeads,usingaBulletBlenderGoldpower-setting10for5min (Next Advance Inc., Averill Park, NY, USA). The homogenized samples were incubated at 95C for 10min and sonicated for 10min.

FFPEtissueswereextractedusingascalpel,deparaffinizedand rehydratedbywashinginxylene(3),andin100%ethanol(2), 96% ethanol (2), 70% ethanol (2), water. The samples were homogenizedin0.5mLlysisbufferwithsteelbeads,usingaBullet BlenderGoldpower-setting10for5min(NextAdvanceInc.,Averill Park,NY,USA).Thehomogenizedsampleswereincubatedat95C for60minforde-crosslinkingformalinfixation,andsonicatedfor 10min[16].

Thetotallysateproteinconcentrationwasdeterminedusinga bicinchoninic acid assay (BCA) for normalization of sample materialwithBSAasstandard,aswell asabsorbanceat280nm (A280) using a NanoDrop 1000 UV–vis Spectrophotometer (ThermoScientific,Waltham,MA,USA).Foreachsample,avolume correspondingto100

m

gproteinwastransferredtoindividual YM-30kDaspinfiltersfordigestion(Millipore,Billerica,MA,USA)and centrifuged.Allcentrifugationstepswereperformedat14,000g for 15min at 4C. Protein disulfide bonds were reduced with 12mM tris(2-carboxyethyl) phosphine (Thermo Scientific, Wal-tham, MA,USA)for 30minat37C,and alkylated with50mM chloroacetamide(Sigma–Aldrich,St.Louis,MO,USA)for20minat 37C,andcentrifugedaftereachstep.Thecysteinealkylationwas doneusingchloroacetamideinsteadofiodoacetamidespecifiedin theoriginal protocol[24]. Theprotocolmodificationwas intro-ducedasspecificityissueshavebeenreportedwithiodoacetamide alkylation, and chloroacetamide has been suggested as an

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alternative [28,29]. The reducing and alkylating agents were dissolvedin120mMSDCin50mMtriethylammonium bicarbon-ate(TEAB),pH8.5.Inpreparationfordigestion,400

m

Ldigestion buffer(12mMSDCin50mMTEAB)wasaddedtothespinfilterand centrifuged.A1:50(w/w)trypsin:proteinratiodissolvedin50

m

L digestionbufferwasaddedtothespinfilter,andthesampleswere digested overnight at 37C. The flow-through containing the peptideswasretrievedbyadditionof50

m

Ldigestionbufferand centrifugation.TofacilitateSDCremoval,aphaseseparationwas performedwith3:1(v/v) ethylacetate:sampleand acidifiedby additionofformicacid(FA)toafinalconcentrationof0.5%.Total phase separation was achieved by 2min agitation followed by centrifugation. The aqueous phase was collected and vacuum centrifugedovernightandthedrypeptideproductwasstoredat

80Cuntiltimeofanalysis. 2.3.Massspectrometryanalysis

Thesampleswereresuspendedin2%acetonitrile(ACN),0.1% FA,brieflysonicated,and5

m

gtotalpeptidematerialwasanalyzed perLC–MSanalysis,inarandomsampleorder[30].Thesamples were analyzed using a UPLC-nanoESI MS/MS setup with an NanoRSLCsystem(Dionex,Sunnyvale,CA,USA).Thesystemwas coupledonline with an emitterfor nanospray ionization (New objectivepicotip360-20-10)toaQExactivePlusmass spectrome-ter(ThermoScientific,Waltham,USA).Thepeptidematerialwas loadedontoa2cmtrappingreversedphaseAcclaimPepMapRSLC C18 column (Dionex), and separated using an analytical 50cm reversedphaseAcclaimPepMapRSLCC18column(Dionex).Both columnswerekeptat40C.Thesamplewaselutedwithagradient of96%solventA(0.1%FA)and4%solventB(0.1%FAinACN),which was increasedto 10% solventBona 1minramp gradient at a constantflowrateof300nL/min.Subsequently,thegradientwas raisedto30%solventB, ona 180minramp gradient.Themass spectrometerwasoperatedinpositivemode,selectingupto12 precursorionswithamasswindowofm/z1.6basedonhighest intensityforHCDfragmenting,atanormalizedcollisionenergyof 27.Selectedprecursorsweredynamicallyexcludedfor fragmenta-tionfor30s.

TheMSproteomics datahavebeendeposited tothe Proteo-meXchangeConsortiumviathePRIDEpartnerrepositorywiththe datasetidentifierPXD002029[31,32].

2.4.Peptidemodificationanalysis

WeconductedaPTManalysiswiththepurposeofidentifying themostcommonlysingleobservedpeptidemodifications,based onthedatafromthecolonbiopsies.Mascotgenericformatfiles

weregeneratedfromtherawdata-filesinProteomeDiscoverer1.4 (ThermoScientific,Waltham,USA).TheMascotgenericformatfiles were searched individually using ProteinPilot 4.5 (Rev. 1656, Paragonalgorithm4.5.0.0[33])(SCIEX,Framingham,USA)against theUniprotHomosapiensreferenceproteome(UP000005640,last modified 2015-01-16, protein count 68,015). The files were searched in “thorough” mode with a focus on biological modificationstoinclude303differentPTMs.Togivea representa-tionoftheglobalPTMdistributionthesearchresultwasanalyzed using ProteinPilot Descriptive Statistics Template version 3.001 (SCIEX) according to manufacturers’ instructions. The statistics template included the first 20,000 peptide spectral matches (PSMs)resultingintheidentificationofpeptideswith<1%local peptidefalsediscoveryrate(FDR).Astricterfilteringsettingthan the standard <5% local peptide FDR was applied for included peptides,toensurethatonlyhighconfidencedatawasincludedin theanalysis.

2.5.Proteinidentificationandquantitationdataanalysis

A label-free relative quantitation analysiswas performed in MaxQuant 1.5.1.2. The rawfiles were searched against the previously mentioned H. sapiens Uniprot database [34,35]. All standardsettingswereemployedwithcarbamidomethyl(C)asa staticpeptidemodification,anddeamidation(NQ),oxidation(M), formylation (N-terminal and K), and protein acetylation (N-terminal) as variable modifications. The output containing the listofproteinsidentifiedbelow1%FDRandtheirabundanceswas further filtered and processed in Perseusv1.5.0.31. Initially, all reversehitsandproteinstaggedascontaminantswereremoved fromfurtheranalysis, andthe datawaslog2-transformed.Two uniquepeptidesormorewasrequiredforaproteinquantitation. Additionally,anon-zeroquantitationvalueinatleasttwoofthesix biopsiesfromminimumonepreservationmethodwasrequiredfor the quantifiable proteins. To characterize proteins unique to a given preservation method, Gene Ontology-annotations were imported from Uniprot knowledgebase when available for all proteins usingSTRAP v1.5[36].Aprinciple componentanalysis (PCA) was performed in Perseus, with all measured protein abundancesasinput.AsPCAdoesnotallowmissingvalues(i.e., proteinswhereaquantitationvaluewasnotobtainedforagiven replicateanalysis),missingvalueswerereplacedwithvaluesfrom anormaldistribution(width0.3anddownshift1.8)tosimulate signals from low abundant proteins [37]. The grouping of the replicatesonthePCAscoresplotswasinvestigated.

To investigate themeasured protein abundances across the different preservation methods, the protein abundances were combinedmethod-wisebythemean.Thedatawasinvestigatedby

Fig.1.Representativetwo-dimensionalLC–MSheatmapsfromtheanalysisofthecolonbiopsiespreservedby(a)directfreezing(DF),(b)RNAlater,or(c)immediate formalin-fixed,paraffin-embedded(iFFPE).

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scatterplotsandPearson’scorrelationcoefficientswerecalculated in Perseus. Protein physicochemical properties were calculated usingProtParamontheExPASyServerforphysicochemicalbias analysis[38].

3.Results

3.1.Peptidemodificationanalysis

All sampleswereanalyzed byLC–MS without contaminants interruptingthestablespray,suchasincompletelyremovedsalts. Wegeneratedandinspected2DLC–MSheatmaps(Fig.1)andno repetitiveintensesignalscouldbeobserved,which wouldhave indicatedremaining detergentsor polymersin the samples.To facilitate the identification of modified peptides and elucidate tentative preservation method specific modifications, we per-formeda peptide PTManalysis of the data from the24 colon biopsies (six from each preservation method) in ProteinPilot, whichincluded303differentmodifications.Theanalysis elucidat-edthepresenceofPTMsonthepeptidelevelfromtheDF,RNAlater, iFFPE, and sFFPE preserved biopsies (Table 1). For all four preservationmethods,atleasthalfofallpeptideswereidentified inamodifiedstate.Asexpected,thelowestnumberofmodified peptideswereidentifiedintheDFpreservedsamples.Asimilar ratio of modified peptides was found in the iFFPE and sFFPE preservedtissues,andthehighestratioofmodifiedpeptideswas foundin theRNAlaterpreservedtissue.FormylatedN-terminals and formylatedlysines (K) were expectedartifacts in theFFPE preservationmethodsastheyareintroducedduringtheformalin fixation. Accordingly, a higher ratio of peptides carrying the modificationswasfoundintheiFFPEandsFFPEpreservedsamples, compared to RNAlater and DF (Table 2). Finally, an increased numberofdeamidatedpeptides(NandQ)wereidentifiedinthe FFPEpreservedbiopsiescomparedtoDForRNAlater.Basedonthe PTManalysis,carbamidomethyl(C),oxidation(M),deamidated(N or Q), formyl (N-term or K) and acetyl (protein N-term) modificationwereincludedinthesubsequentdataanalysis. 3.2.Preparationmethodspecificproteins

TheLC–MSraw-datafromthecolonbiopsieswereprocessedin MaxQuant to identify and quantify proteins. A similar mean numberofproteinswereidentifiedfromthebiopsiespreservedby DFandRNAlater,namely3840and3718proteins(Fig.2).Biopsies preservedby iFFPE and sFFPE yielded a statistically significant lowernumberofidentifiedproteins(p<0.05)asdeterminedbya two-sample t-tests, compared to DF and RNAlater, namely 3384 and 3328 proteins. The difference in mean number of identifiedproteinsbetweeniFFPEandsFFPEwasminorandnot foundtobestatisticallysignificant(p>0.05).Thisindicatedthat the30minstored prior tostabilizationof thesFFPE preserved colonbiopsiesdidnotresultinproteindegradationtoanextend thathadanimpactonthenumberofidentifiableproteins.

The mean protein abundances were calculated for each preservation method, and the overlap between quantifiable proteinswereinvestigated(Fig.3).Ahighernumberofproteins were quantifiable in biopsies preserved by DF and RNAlater comparedtoiFFPE,and5.9%(202)oftheproteinswerenotfound withiFFPE.However,90.2%(3072)ofallquantifiableproteinswere foundusingeithermethod.

We next investigated if any of the methods systematically enrichedorcategoricallylostspecificproteingroups,basedonthe proteins uniquetoany methods. Allquantifiableproteins were classifiedbysubcellularlocationusingavailabledatafromUniprot KnowledgebaseGene Ontology (Fig.4).We chooseto compare proteins,whichwereuniquelyquantifiableintheiFFPEpreserved biopsies,tothecombinedlistofproteinsuniqueintheRNAlateror theDFpreservedbiopsies.RNAlaterandDFuniqueproteinswere combinedintoonegroupasthelistsofquantifiedproteinsusing the two methods werenearly identical sharinga 96.8% (3274) overlap.Theanalysisrevealedonlyminordifferencesinsubcellular locationoftheproteinsuniquetoDFandRNAlatercomparedtoall proteins quantified in the DF preserved biopsies. Biopsies preservedbyiFFPEappearedtobeenrichedforERandnucleus proteins,comparedtotheDFpreservedbiopsies.However,only 23proteinswereuniquetotheiFFPEpreservedbiopsies,and98.7% (3114)oftheproteinsquantifiediniFFPEwerealsoquantifiedin thebiopsiespreservedbytheDFpreservedbiopsiesaswell,sothe differencesareminor.

Wenextinvestigatedthemolecularweightandisoelectricpoint ofthemethodspecificproteins.Themeanmolecularweightand standard deviation (s) were68,183Da (s=85,398Da) for all DF proteins,64,950Da (s=59957Da)forproteins uniquetoDFand RNAlater,and92,626Da(s=78,930Da)foriFFPEuniqueproteins. The23iFFPEuniqueproteinshaveahighermeanmolecularweight thantheDFproteins,but,thevarianceislikewisehigher.Likewise, themeanisoelectricpointsoftheproteinswas6.72(s=1.63)forall DFproteins,6.71(s=1.59)forDFandRNAlateruniqueproteins,and 6.89 (s=1.81) for iFFPE unique proteins. As such, only minor differences couldbe detected in themolecularweight and the isoelectric point between the proteins unique to any method comparedtoallDFproteins.

3.3.Principlecomponentanalysis

WeperformedaPCAtoinvestigatehowthemeasuredprotein abundances vary betweenthe differentlypreserved biopsies.A PCAis astatisticalanalysistechniquethatallowsforreducinga largenumberofvariablestoasmallernumberofgroups(principle components).Thedatacanbevisualizedonscoresplotsbasedon the principle components, and e.g., be used to interpret how samplesinadatasetareseparated/groupedbasedonthevariance ofallmeasuredproteinabundances.Ineffect,aPCAcanbeusedto interpretthevarianceinahighlycomplexdataset,suchasahigh throughputproteomicsdataset[27,39].

Table1

Peptideproperties.Analysisofthefirst20,000peptidespectralmatches(PSMs)resultingintheidentificationofpeptideswith<1%localpeptidefalsediscoveryrate(resulting peptideconfidencelisted).DF:directlyfrozenbiopsies,RNAlater:biopsiespreserveddirectlyinRNAlater,iFFPE:immediateformalin-fixed,paraffin-embeddedbiopsies, sFFPE:biopsiesstoredfor30minpriortoformalin-fixation,paraffin-embedding.Theterminioftheidentifiedpeptidesisgiven,withexpectingterminibeingtryptic. CarbamidomethylatedcysteinesarenotcountedasaPTM,asthemodificationisdeliberatelyintroducedpriortodigestionwithtrypsin.Standarddeviationsaregiven(s).

DF(%) RNAlater(%) iFFPE(%) sFFPE(%)

Unmodifiedpeptides 55.52.5 56.00.6 50.73.8 49.92.2

Modifiedpeptides 44.52.5 44.00.6 49.33.8 50.12.2

Peptideconfidence 97.70.39 98.00.25 98.10.3 98.00.3

Tryptictermini 95.00.4 94.90.4 95.50.5 95.80.3

Semi-specific(onlyonetrypticterminus) 5.00.4 5.00.4 4.40.5 4.10.3

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Allmeasuredproteinabundancesinallcolonbiopsysamples wereusedasinputforthePCA,andatwo-dimensionalscoresplot wasconstructed(Fig.5a).Ascoresplotdescribehowthebiopsies

grouprelativetooneanother,basedonthedifferencesinmeasured protein abundances of all proteins. Biopsies in which similar proteinabundanceshavebeenmeasuredwillbecloseinspaceon thescoresplot,relativetotheotherbiopsies.Principalcomponent 1 and principle component 2 representthe largestand second largestvarianceintheproteinabundancedataset,respectively,and explained34.4%ofthevarianceintheproteinabundanceinthis dataset.Inallcases,thebiopsiesfromtheindividualparticipants groupedtogether.Principalcomponent1mainlyseparatedtheDF andRNAlaterpreservedbiopsiesfromtheFFPEpreservedbiopsies, whereas principle component 2 mainly separated the two participantsfromwhichthebiopsiesoriginated.

Theimpactofeachproteinontheseparationofthebiopsieson thescoresplot,canbevisualizedona loadingsplot.Tofurther identify the main differences in the dataset leading to the separation of DF and RNAlater stabilized samples and FFPE samples,theloadingplotofprinciplecomponent1was investi-gated. The isoelectric point and molecular weight of proteins having a loadings score greater than 0.4 were investigated (Fig.5b,blueandredcircles).Themeanmolecularweightforthe 107proteinswithgreaterthan0.4loadingscorewas34,414Da,and 55,226Daforthe15proteinwithlessthan 0.4loadingscore.The meanmolecularweightofallDFproteinswas68,183Da.Likewise,

Table2

Residueandterminalspecificmodifications.Method-visepooledtoptenpeptidemodificationsinbiopsiespreservedbydirectfreezing(DF),RNAlater,immediate formalin-fixed,paraffin-embedded(iFFPE),or30minstoredformalin-fixed,paraffin-embedded(sFFPE).Modifiedaminoacidisgivenbyone-lettercode.Thefirst20,000peptide spectralmatches(PSMs)resultingintheidentificationofpeptideswith<1%localpeptidefalsediscoveryratewereincludedintheanalysis.PTMpeptidesofpossibleisthe percentageofpeptidesfoundinagivenmodifiedstate,inrelationtoallpeptideswhichcouldhavethespecificmodification.Standarddeviationsaregiven(s).

Feature Dmass(Da) PTMpeptidesofpossible

DF(%) RNAlater(%) iFFPE(%) sFFPE(%)

Carbamidomethyl(C) 57.0215 1000.0 1000.0 1000.0 1000.0

Oxidation(M) 15.9949 29.33.9 33.71.8 45.412.3 49.716.9

Proteinterminalacetyl@N-term 42.0106 34.44.7 34.12.1 39.03.0 39.22.9%

Gln->pyro-Glu@N-term 17.0265 27.06.4 24.02.2 29.01.8 29.04.6% Deamidated(N) 0.984 11.70.5 11.80.5 17.01.0 16.00.8% Deamidated(Q) 0.984 6.20.1 6.20.1 7.60.4 7.40.5% Formyl@N-term 27.9949 5.13.6 2.80.6 6.42.4 7.33.2% Oxidation(P) 15.9949 1.30.3 1.10.6 1.80.3 1.70.4% Dioxidation(M) 31.9898 0.70.2 0.80.1 1.50.6 1.60.8 Met->Hcy(M) 14.0157 0.60.1 0.60.1 0.40.1 0.40.1 Formyl(K) 27.9949 0.40.5 0.20.1 3.70.5 3.30.4% Methyl(K) 14.0157 0.00.0 0.00.0 2.50.3 2.30.3% + + + + 3200 3400 3600 3800 4000

DF RNAlater iFFPE sFFPE

p < 0.0005 p = NS

p < 0.0005 p = NS

Protein IDs

Fig.2.Numberofidentifiedproteinsintheindividualcolonbiopsies,usingthe direct freezing (DF), RNAlater, immediate formalin-fixed, paraffin-embedded (iFFPE),and30minstoredformalin-fixed,paraffin-embedded(sFFPE)preparation protocols.Significantchangesdetectedbytwo-samplet-testsandrepresentedby p-values.NS:notsignificant,p<0.05wereconsideredsignificant.

Fig.3.Numberofproteinsuniquelyquantifiedinthecolonbiopsiespreservedby(a)directfreezing(DF),RNAlater,orimmediateformalin-fixed,paraffin-embedded(iFFPE), and(b)iFFPEor30minstoredformalin-fixed,paraffin-embedded(sFFPE)preservationprotocols.

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themeanisoelectricpointofproteinswithgreaterthan0.4loading scorewas6.52(s=1.69),andforproteinwithlessthan 0.4was 8.14(s=2.80),comparedto6.72(s=1.63)forallDFproteins.

Wenextinvestigatedtheproteinsmainlycontributingtothe separationofthetwoparticipant.Inourdatasetthiswasbasedon PCAprinciple component 2.The twoproteins withthehighest impact wereHLAclass I histocompatibility antigen A-23alpha chain(HLA-A)andHLAclassIIhistocompatibilityantigen DRB1-4betachain(HLA-DRB1).Botharewelldescribedintheliterature andknowntoexistinhighgeneticdiversity.Toensurethatthetwo proteinswererepresentedsimilarlyusingalltissuepreservation methods,wecalculatedthemeanfoldabundancechangebetween participantAandB,ofHLA-DRB1foreachmethod.HLA-DRB1was foundinallbiopsiesandthefoldchangeoftheproteinbetween participant B and A was 5.00in DF biopsies, 4.32in RNAlater biopsies,5.66iniFFPEbiopsies,and5.55insFFPEbiopsies.HLA-A was found in all biopsies from participant A and none from participantB, regardless ofmethodofsample stabilization.The sensitivity of the assay thereby seems retained regardless of methodofsamplestabilizationfortheseproteins.

3.4.Proteinabundancescatterplots

Toinvestigatepreservationmethodinducedvariationsacross allproteinabundances,weinvestigatedscatterplotsoftheprotein abundances.Scatterplotscomparethequantitativevalueofevery proteinbetweentwopreservationmethods,plottingtheprotein abundanceofdifferentmethodsonthexandy-axis,respectively. Ideally, the different tissue preservation methods should yield identicalproteinabundances,representedbyaPearson’s correla-tion coefficient of one, indicating a perfect correlation. The measured protein abundance in the three biological replicates were combined preservation method wise by the mean, and scatterplotsweregeneratedcomparingtheRNAlater, iFFPE,and sFFPEpreservationmethodstoDF,aswellasiFFPEtosFFPE(Fig.6). The lowest correlation coefficient (0.943) was calculated comparing DF preserved tissue to sFFPE preserved tissue. The highest correlation (0.987) was found between the DF and RNAlaterpreservedtissues,which wassimilartothecoefficient betweentheiFFPEandsFFPE(0.983)proteinquantitations.Inall cases, the Pearson’s correlation coefficients were close to one, indicatingagoodcorrelation.

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Fig.4.ComparativecellularcompartmentGeneOntologyannotationofquantifiedproteinsinthecolonbiopsiestoinvestigatepotentialmethodbiasestowardspecific proteintypes.Top-bar:allquantifiableproteinsinthedirectlyfrozen(DF)biopsies;middle-bar:proteinsuniquelyquantifiedintheDFandRNAlaterpreservedbiopsies; lower-bar:proteinsuniquelyquantifiableintheimmediateformalin-fixed,paraffin-embedded(iFFPE)biopsies.Theannotationshavebeennormalizedto100%,andnumber ofincludedproteinsaregivenforeachpreservationmethod.

-20 -10 0 1 0 2 0 Principle Component 2 (9.4%) -30 -20 -10 0 10 20 30 Principle Component 1 (26.8%) DF and RNAlater

iFFPE and sFFPE A

A B B

a)

b)

Loading... -1 0 1 Component 2 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 Component 1 MUC1 PTMA GC UGT2B17 APOA2 ORM1 COL4A2 HLA-DRB1 LMOD1 HLA-A GSTT1 RPS11

Fig.5. Principlecomponentanalysis(PCA)scoresplotandloadingsplotbasedon principlecomponent 1and 2,with allprotein abundances in thedifferently preservedcolonbiopsiesfromtwoparticipantsasinput.(a)Scoresplotofcolon biopsiesfromparticipantA(filledsymbols)andparticipantB(hollowsymbols) werestabilizedbydirectfrozen(DF)(&and&),RNAlater( and ),immediate formalin-fixed,paraffin-embedded(iFFPE)( and ),or30minstored formalin-fixed,paraffin-embedded(sFFPE)( and )preservationprotocol.(b)Loadingsplot withallquantifiableproteins.Proteinswith0.4wereanalyzed( and )were chosenforfurtheranalysis.Thegenenamesofthetopthreeextremeproteinson eachaxisaregiven.(Forinterpretationofthereferencestocolorinthetext,the readerisreferredtothewebversionofthisarticle.)

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ThehighPearson’s correlationcoefficientbetweeniFFPEand sFFPE indicates that delaying tissue biopsy stabilization with 30minonlyhadaminorimpactonthemeasuredglobalprotein abundances.TofurtherinvestigatethedifferencesbetweeniFFPE andsFFPEandtheimpactofthedelayedsamplestabilization,we addedproteininstabilityindexscoresfromExPASyforchanging proteins[38].Theinstabilityindexscoreprovidesanestimationof aproteins’invivostability[40].Aninstabilityindexscorebelow 40 estimates the protein to be stable [41]. We assumed that proteinspronetoinvivodegradation,wouldbepartlydegradedin the30mindelayinstabilizationinthesFFPEpreservedbiopsies. Therefore,wechoosetoinvestigateproteinswithagreaterthan twofoldabundancedecreaseinsFFPEbiopsiescomparedtoiFFPE. Thiscameto48proteinsand113iFFPEuniqueproteins,andthe mean protein instability index score for these proteins was calculated tobe42.0 (s=13.3). Thescore was compared tothe mean protein instability index score for proteins with similar abundancesbetweensFFPEandiFFPE, whichweassumedtobe moreinvivostable.Wechoosetoincludeallproteinswithlessthan 0.05meanfoldchangedifferencebetweeniFFPEandsFFPE(fold change1.05–0.95).Thiscameto677proteinswithameanprotein instability index score 43.8 (s=10.7). Considering the standard deviation, the two stability indexes are highly similar. We additionally investigated the molecular weight and isoelectric pointoftheseproteingroups,andsimilarpropertieswerefound (datanotshown).Thisindicatesthattheproteininstabilityindex scoreisoelectricpointandmolecularweightcannotaccountfor theabundancechangeofthe161proteins(Supplementarylist2) foundwithlowerabundanceinthesFFPEbiopsies.

4.Discussion

We examined the impact of sample preservation on a discovery-based proteome analysis, in order to enable reliable proteomeanalysisofdifferentlypreservedsamples.Humancolon mucosa biopsies were extractedand immediately preserved in RNAlater orby FFPE.Tosimulate aclinical scenario,one setof biopsieswerestoredfor30min(sFFPE)atambienttemperature before sample stabilization with formalin. The samples were comparedtosnapfrozenbiopsies(DF),wheretheintroductionof chemicalartifacts,whichmightinterferewithaproteomeanalysis, is minimal. The main finding of the study was that biological samplescanbestoredinRNAlaterandpreservedbyFFPEwitha minimalimpactontheresultofaquantitativeproteomeanalysis compared to DF preserved samples. Additionally, delaying the formalinsamplestabilizationfor30minonlyhadaminorimpact inourdataset.Similarresultsandconclusionsregardingpathway regulations can bereached for thesamplesas compared toDF preservedsamples.EspeciallyRNAlaterpreservationwasfoundto beapromisingalternativetosnapfreezingsamplesforproteomic studies.

We performed a peptide PTM analysis to investigate the efficiency of the de-crosslinking protocoland toidentify mod-ifications introduced by the preservation methods. The PTM analysisrevealedthatFFPEpreservationofcolontissuecausedan increase in the overall number of peptides identified with a modificationcomparedtoDFandRNAlater.Theanalysisofspecific peptidemodificationsrevealedanincreasedratioofformylated peptides(K-andN-term)intheFFPEpreservedbiopsiescompared

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DF

DF

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iFFPE

sFFPE

a)

b)

d)

DF

iFFPE

sFFPE

c)

r = 0.987

r = 0.950

r = 0.943

r = 0.983

Fig.6.Scatterplotsofthelog2transformedproteinabundancesofallquantifiableproteinsinthecolonbiopsies,combinedbythemeanpreservationmethodwise.The proteinabundancesareplottedagainstoneanotheronthex-andy-axes,respectively.Correlationsbetweenmeasuredproteinabundancesusingthedifferentmethodsare representedbyPearson’scorrelationcoefficients(r),wherer=1signifiesaperfectcorrelation.Proteinabundancesindirectlyfrozen(DF)colonbiopsieswascomparedto(a) RNAlater,(b)immediateformalin-fixed,paraffin-embedded(iFFPE),or(c)30minstoredformalin-fixed,paraffin-embedded(sFFPE),and(d)iFFPEandsFFPEarecompared.In allcasesr-values>0.94werecalculatedindicatingagoodcorrelation.Linesindicatesafourfoldproteinabundancedifferencein(a–c),andatwofoldproteinabundance differencein(d).

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toRNAlaterandDF.ThiswasanexpectedartifactfromtheFFPE crosslinking. Additionally, an increased number of methylated lysine-containingpeptides was identifiedin theFFPEpreserved samples,inaccordancewithfindingsinarecentstudywhichgives validationtotheappliedmethodology[42].However,lessthan10% ofthePSMsresultedinidentificationofpeptideswithaformyl modification,indicatingthattheappliedde-crosslinkingprotocol iseffective.Anincreasednumber of deamidatedpeptides were identifiedinbiopsiespreservedwiththeFFPEprotocolscompared tothe DF and RNAlater protocols. The modification was likely introduced during the exposure to non-physiological pH and elevated temperatures during the sample preparation (alkaline pH)whichwasprolongedintheFFPEprotocol[43,44].Onlyminor differencescouldbeobservedinthePTMfrequenciesbetweenDF andRNAlaterstabilizedtissue,indicatingthattheintroductionof identifiablechemicalartifactswiththetwomethodsissimilar.

The alkylationof cysteine-containing peptideswith chloroa-cetamide was demonstrated, and 100% of cysteine-residue containingpeptideswerefoundinanalkylatedstate.Additionally, nopeptidealkylationartifactswereobservedinthisstudywith frequenciesof more than 0.6% of possibly modifiablepeptides, such as carbamidomethylated N-terminal and non-cysteine residues.In a recent study of porcine synovial fluid using the FASPprotocolwithiodoacetamidealkylation,2.7%ofallpeptides wereidentifiedwithacarbamidomethylatedN-terminal,and1.9% of thecysteine- and lysine-containing peptides wereidentified withancarbamidomethylatedlysine[45].Theanalyzedsamplesin thementionedstudyareverydifferentfromthesamplesanalyzed inthisstudy.Nonetheless,thecomparisonpointstoanimproved alkylationspecificitywhenusingtheprotocolwith chloroaceta-midecomparedtoiodoacetamide,inagreementwithaprevious study[29].

The differences in frequencies of the various modifications demonstratethatthemajorityofthePTMsareintroducedduring thesamplepreservation.Assuch,thePTM-tablecanbeusedto chooserelevantmodificationsfordatabasesearching.

Wewereabletoidentifyasimilarnumberofproteinsintissue preserved by DF and RNAlater. Other proteomic studies have reported a similar or slightly increased number of proteins identified from bacteria, yeast, and fish preserved in RNAlater comparedtoDF,inagreementwithourfindingsonhumantissue

[5,7,9].Weidentifiedastatisticallysignificantlylowernumberof proteinsintheFFPEpreservedtissuecomparedtoDFandRNAlater preservedbiopsies.These findingsare in agreement withwhat otherproteomicstudiesonhumansampleshavereported[2,3, 14–21].Eventhoughthenumberofquantifiedproteinswaslower intheFFPEbiopsies,92.3%of theproteins quantifiedintheDF preservedbiopsies werealso quantified in the FFPEpreserved biopsies. The high overlap demonstrates the feasibility of conductingproteomicsonFFPEsamples.

WeconductedaGeneOntology-basedanalysisofproteinsonly foundintheiFFPEpreservedbiopsies,comparedtotheRNAlater and DF preserved. This was done to investigate if the unique proteinsoriginatedfromaspecificcellularlocation.Theanalysis revealedthatproteinsuniquetoagivenmethoddidnotbelongtoa specificsubcellularlocation.Theanalysisoftheisoelectricpoint andmolecularweightoftheuniqueproteingroupsalsodidnot reveal any determining differences. This indicates that no systematiclossorenrichmentofproteinsfromaspecificcellular location, nor proteins with specific physiological properties, is takingplaceusingeitherofthepreservationmethods,andthusan unbiasedanalysisregardlessofsamplepreservationmethod.

The label-free relativequantitation allowed usto assessthe impactofthebiopsypreservationontheproteinabundances.The PCAscores plotof principle component 1 and 2 described the largestvarianceinproteinabundances.ThePCAscoresplotdidnot

separate DFandRNAlaterpreservedcolon tissue,northecolon tissuepreservedbyiFFPEandsFFPE.Thisdemonstratesthatthe biological variance in the measured proteome of the two participants is greater than the variance introduced by the preservationinDFandRNAlater,aswellasiFFPEandsFFPEtissue, respectively.SamplespreservedbyRNAlatercanthusbecompared to sample preserved by DF, as the introduction of proteome changes is minimal. Thesame canbe saidfor iFFPE and sFFPE preserved samples. It should be emphasized that the colon biopsiesoriginatedfromtwoparticipantswithout gastroentero-logicalfindings.Thedifferenceinproteinabundancesbetweenthe twosetsofbiopsiescan,therefore,beexpectedtobelessthanwhat is measured in disease studies, e.g., of inflammed and non-inflammed colon biopsies [27]. Even so, we were still able to separatebiopsiesfromthetwoparticipants.Thefindingsfromthe PCAaresupportedbythescatterplots,whereproteinabundances across thedifferent methods were investigated.The calculated Pearson’scorrelationcoefficientsofRNAlater,iFFPE,sFFPE,andDF preserved colon biopsies were all greater than 0.94. This demonstratesthegoodcorrelationinmeasuredprotein abundan-cesbetweenthedifferentmethods,i.e.,alowimpactofthemethod of preservation on the overall measured protein abundances

[27,46].Theresultindicatesthatthelargestdifferenceisbetween theDFandFFPEmethods,inagreementwiththePCA.

Additionally,theslightlybettercorrelationofDFandRNAlater preservedsamplescomparedtoiFFPEandsFFPE,indicatesthatthe impactontheproteomeoftheRNAlatertreatmentislessthanthe impact of a 30min storage at room temperature.As RNAlater samplesarestoredat24hatroomtemperaturepriortostorageat 80C, the result indicatesthat RNAlaterefficientlyinhibits in vivobiologicalactivityinthebiopsies.Thesimilarrelativeamounts measured using either method, demonstrates that all four preservation methods can be used tostabilize tissues prior to proteomic analysis. This is supported by the PCA showing the ability toseparate theparticipantsbygenomicdiverseproteins suchasthetwoHLAproteinclasses.

Thecollectionofsamplesforresearchathospitalsandclinics shouldnotinterferewiththestandard clinicalprotocols,which meansthatadelaybeforeasamplecanbestabilizedisinmany casesunavoidable.Ithasbeenreportedthattheaveragetimefrom specimen extraction to processing in a surgical department is 19.3min[4].Thetimecanbeexpectedtovarybetweendifferent hospitals/departments.Therefore,wechosetostoresamplesfor 30minpriortoformalinstabilization(sFFPE).Wedidnotfinda significant decreasein thenumber ofproteins wewereableto recoverfromthesFFPEsamplescomparedtoiFFPE.Nordidwefind vastchangesintheoverallproteinabundancesbyscatterplots,nor changesintheproteinstabilityindexscorescausedbythe30min delayed sample stabilization. Our findings thereby strongly supportthat clinically obtainedtissue biopsiescan beusedfor quantativeproteomicsresearch,evenwhenstabilizationhasbeen delayed.Wedidnotfocusouranalysisontheimpactofunstable proteinmodifications,suchasproteinphosphorylationorproteins withshortinvivolifetime/stability.Severalstudieshavereported changesinthephosphorylation-patternsofseveralstress-related proteins following tissue extraction, when sample stabilization was delayed even a few minutes [4,47,48]. Clinical samples obtainedwithadelaymight,therefore,constitutesuitablematerial for global quantative proteome research focusing on protein abundances,butbepoorlysuitablefore.g.,proteinphosphoprotein studies.Wehaveincludedthelistof48proteinswithagreaterthan two-fold abundances difference between iFFPE and sFFPE pre-servedbiopsiesandthe113iFFPEuniqueproteins(Supplementary Table2).Itispossiblethattheseproteinsdisplayalowerinvivo stabilitythanrepresentedbytheproteininstabilityindexscores.

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OtherstudieshavedemonstratedthatRNAlatercanbeusedto stabilize bacteria, fungus, and human cervical swabs prior to proteomeanalysis [5,8,9]. Combined withthefindings in other studiesperformedonbacteriaandyeast,ourfindingsdemonstrate thatRNAlatercanbeusedforuniformpreservationofawiderange of biological material prior to proteomic, transcriptomic, and genomic studies. The need for snap freezing samples can be reduced,whichcanbeimpracticalespeciallyinclinicalsituations. Additionally,RNAlaterandFFPEsamplepreservationremovesthe needforquicksamplehandling.ThisisincontrasttoDFpreserved tissuewheretheprotectiveeffectoffreezingonlypersistsuntilthe sample is thawed, making a rapid sample handling prior to proteaseinhibitioncritical[5].Thepossibilitytoutilizeestablished biobanksforproteomeanalysisconstitutesavastsourceof well-characterizedbiologicalmaterialforclinicalproteomicsresearch. 5.Conclusion

ThisisthefirststudytoinvestigatetheimpactofRNAlaterand FFPE stabilization of human tissues on PTMs and protein quantitation. Using 24 humancolon biopsies from two partic-ipants,wehavedemonstratedthathumantissuesamplescanbe stabilizedand preservedby RNAlateror FFPEasalternatives to snapfreezing with a minimal impactonthe qualityof protein quantifications.EspeciallyRNAlaterpreservationwasfoundtobea promising alternative to snap freezing samples for proteomic studies. Comparable proteomics pathway information can be extractedfromtissuepreservedwitheithermethod.Additionally, delayingtissuesamplestabilizationwithformalinfor 30minto simulateaclinicalsituation,onlyresultedinaminorimpactonthe qualityoftheproteinquantitations.Ourfindingsthereby demon-stratethatbiobankscontainingRNAlaterpreservedsamples,FFPE preserved samples, and samples obtained with a delay in stabilization,can be used for proteomeanalysis. Similar result andconclusionscanbeobtainedontheglobalproteomelevelas whenstudyingsnapfrozensamples.Thesuggestedprotocolscan thusbeused,e.g.toprovideretrospectiveinformationconcerning diagnosis,responsetotherapyandnoveldrugdiscovery. Conflictofinterest

VibekeAndersen receives compensationasa consultantand advisoryboardmemberforMSD(Merck)andJanssen.

Supportinginformation

SupplementaryTable1containsthefulllistofPTMsforeach biopsy. Supplementary Table 2 contains the list of proteins displayingagreaterthan2foldchangebetweeniFFPEandsFFPE. TheMSproteomicsdatahavebeendepositedtothe ProteomeX-change Consortium via the PRIDE partner repository with the datasetidentifierPXD002029[31,32].Bennikeetal.[49]contains anexpandeddescriptionofalldepositedproteomicsdatafilesand SupplementaryTable1in[49]containsthefulllistofidentified proteins(<1%FDR)inthehumancolonbiopsies.

Acknowledgements

Wewouldliketothankthetwoparticipantsfortakingpartin thestudy.DepartmentofPathologyatAalborgUniversityHospital, Denmark is acknowledged for performing the FFPE sample preservation.KnudandEdithEriksensMemorialFoundationand Ferringareacknowledgedforgrants,enablingthecollectionofthe biological sample material (VA grant). The Obelske family foundation, the Svend Andersen Foundation and the SparNord foundationareacknowledgedforgrantstotheanalyticalplatform,

enabling this study (AS grants).The Lundbeck Foundation and CarlsbergFoundation areacknowledged forgrantsenabling the analysis(TBBgrants).ThestaffattheHospitalofSouthernJutland arethankedforexcellenttechnicalassistance.Finally,theauthors wouldliketothankthePRIDEteamformakingtheproteomicsdata publicallyavailable.

AppendixA.Supplementarydata

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. euprot.2015.10.001.

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