LC–MS/MS-based quantification of efflux transporter proteins at the BBB

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LC–MS/MS-based quantification of efflux transporter

proteins at the BBB

David Gomez-Zepeda, Méryam Taghi, Maria Smirnova, Philippe Sergent,

Wang-Qing Liu, Cerina Chhuon, Michel Vidal, Martin Picard, Elizabeth

Thioulouse, Isabelle Broutin, et al.

To cite this version:

David Gomez-Zepeda, Méryam Taghi, Maria Smirnova, Philippe Sergent, Wang-Qing Liu, et al.. LC–

MS/MS-based quantification of efflux transporter proteins at the BBB. Journal of Pharmaceutical and

Biomedical Analysis, Elsevier, 2019, 164, pp.496-508. �10.1016/j.jpba.2018.11.013�. �hal-02350085�

Contents lists available atScienceDirect

Journal

of

Pharmaceutical

and

Biomedical

Analysis

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a

LC–MS/MS-based

quantification

of

efflux

transporter

proteins

at

the

BBB

David

Gomez-Zepeda

,

Meryam

Taghi

,

Maria

Smirnova

,

Philippe

Sergent

,

Wang-Qing

Liu

,

Cerina

Chhuon

,

Michel

Vidal

,

Martin

Picard

,

Elizabeth

Thioulouse

_,

_Isabelle

_Broutin

,

Ida-Chiara

Guerrera

,

Jean-Michel

Scherrmann

,

Yannick

Parmentier

,

Xavier

Decleves

,

Marie-Claude

Menet

a_{Inserm,UMR-S1144,ResponseVariabilitytoPsychotropics,Paris,France} b_{UniversitéParisDescartes,Paris,France}

c_{UniversitéParisDiderot,Paris,France}

d_{TechnologieServier,Départementderecherchebiopharmaceutique,Orléans,France}

e_{CNRS,UMR8638,ChimieOrganique,MédicinaleetExtractiveetToxicologieExpérimentale,Paris,France} f_{PlateformeProtéomique3P5-Necker,SFRNecker,US24,UniversitéParisDescartes,Paris,France} g_{UFBiologiedumédicamentettoxicologie,HôpitalCochin,APHP,Paris,France}

h_{CNRS,UMR8015,LaboratoiredecristallographieetRMNbiologiques,France} i_{Servicedebiochimie,HôpitalArmandTrousseau,APHP,Paris,France} j_{UFHormonologie,HôpitalCochin,APHP,Paris,France}

a

r

t

i

c

l

e

i

n

f

o

Received26March2018

Receivedinrevisedform29October2018 Accepted5November2018

Availableonline8November2018 Keywords: ABCtransporters LC–MS/MS MRMquantification Targetedproteomics

a

b

s

t

r

a

c

t

Targetedproteinquantificationusingtandemmassspectrometrycoupledtohighperformance

chro-matography(LC–MS/MS)hasbeenusedtoquantifyproteinsinvolvedintheabsorption,distribution,

metabolismandexcretion(ADME)ofxenobioticstobetterunderstandtheseprocesses.Atthe

blood-brainbarrier(BBB),theseproteinsareparticularlyimportantforthemaintenanceofbrainhomeostasis,

butalsoregulatethedistributionoftherapeuticdrugs.Absolutequantification(AQUA)isachievedby

usingstableisotopelabeledsurrogatepeptidesspecifictothetargetproteinandanalyzingthedigested

proteinsinatriple-quadrupolemassspectrometerinmultiplereactionmonitoring(MRM)modeto

achieveahighspecificity,sensitivity,accuracyandreproducibility.Themainobjectiveinthisworkwas

todevelopandvalidateanUHPLC-MS/MSmethodforquantificationoftheATP-bindingcassette(ABC)

transporterproteinsBcrpandP-gpandNa+/K+ATPasepumpattheBBB.Threeisoformsofthe␣-subunit

fromthispump(Atp1a1,2and3)werequantifiedtoevaluatethepresenceofnon-endothelialcells

intheBBBusingonecommonandthreeisoform-specificpeptides;whileBcrpadP-gpwere

quanti-fiedusing2and3peptides,respectively,toimprovetheconfidenceontheirquantification.Theprotein

digestionwasoptimized,andtheanalyticalmethodwascomprehensivelyvalidatedaccordingtothe

AmericanFoodandDrugAdministrationBioanalyticalMethodValidationGuidancepublishedin2018.

Linearityacrossfourmagnitudeorders(0.125to510pmol·mL−1_{)sub-pmol·mL}−1_LODandLOQ,

accu-racyandprecision(deviation<15%andCV<15%)wereprovenformostofthepeptidesbyanalyzing

calibrationcurvesandfourlevelsofqualitycontrolsinbothapuresolutionandacomplexmatrixof

digestedyeastproteins,tomimicthematrixeffect.Inaddition,digestionperformanceandstability

ofthepeptideswasshownusingstandardpeptidesspikedinayeastdigestormousekidneyplasma

membraneproteinsasastudycase.Thevalidatedmethodwas usedtocharacterizemousekidney

plasmamembraneproteins,mousebraincorticalvesselsandratbraincorticalmicrovessels.Mostof

theresultsagreewithpreviouslyreportedvalues,althoughsomedifferencesareseenduetodifferent

sampletreatment,heterogeneityofthesampleorpeptideused.Importantly,theuseofthreepeptides

Abbreviations:ADME,absorption,distribution,metabolismandexcretion;BBB,blood-brain;MRM,multiplereactionmonitoring;SIL,stableisotopelabeled. ∗ Correspondingauthorsat:Inserm,UMR-S1144,ResponseVariabilitytoPsychotropics,Paris,France.

E-mailaddresses:david.gomez.zepeda@cinvestav.mx(D.Gomez-Zepeda),marie-claude.menet@parisdescartes.fr(M.-C.Menet).

1 _{Currentaddress:LaboratoiredeBiologiePhysico-ChimiquedesProtéinesMembranaires,CNRSUMR7099,InstitutdeBiologiePhysico-Chimique(IBPC),Paris,France.} https://doi.org/10.1016/j.jpba.2018.11.013

allowedthequantificationofP-gpinmousekidneyplasmamembraneproteinswhichwasbelowthe

limitofquantificationofthepreviouslyNTTGALTTRpeptide.Thedifferentlevelsobtainedforeach

pep-tidehighlighttheimportanceanddifficultyofchoosingsurrogatepeptidesforproteinquantification.In

addition,usingisoform-specificpeptidesforthequantificationoftheNa+/K+ATPasepump,weevaluated

thepresenceofneuronalandglialcellsonratandmousebraincorticalvesselsinadditiontoendothelial

cells.Inmouseliverandkidney,onlythealpha-1isoformwasdetected.

©2018ElsevierB.V.Allrightsreserved.

1. Introduction

Absolutequantificationofproteinsbymassspectrometry(MS) isusedinmanyfieldsofbiologytocharacterizecellsandtissuesas wellastheirbiologicalprocessesandproteinlevelchanges.These methods havegained popularitybecauseof itshighsensitivity, accuracy,reproducibilityandthepossibilityofanalyzingmultiple proteinsinasingleanalysis.Inbiopharmaceuticalresearch,thereis aparticularinterestintheuseofthesemethodstoquantifyproteins involvedintheabsorption,distribution,metabolismandexcretion (ADME)ofxenobioticsin ordertobetterunderstandthese pro-cesses.Therefore,targetedabsoluteproteinquantificationhasbeen usedtocharacterizetheexpressionoftransporters,receptors,tight junctionproteinsanddrugmetabolizingenzymesindifferentstudy models(invivo[1,2]andinvitro[3]),butalsoinhumansamples [4–7].

The ATP-binding (ABC) transporters family is particularly importantfortheADMEofnutrientsandexogenoussubstances. Theyaremostlylocatedattheplasmamembraneinthetissue inter-facessuchasendotheliaandblood-tissuebarrierswheretheyefflux ahighvarietyofsubstrates.Theyarefoundintheintestine,liver, kidney,heart,lungs,brain,placentaandtestis.TheABCtransporters arethusfundamentalforthemaintenanceofseveralphysiological functionssuchasprotectionfromtoxic substances,transportof importantmetabolitesandcellsignaling.Attheendothelialcells oftheblood brainbarrier(BBB),thesetransportersare particu-larlyimportanttomaintainthebrainhomeostasisbyregulating thepenetrationofdangeroussubstances butalsooftherapeutic drugsintothebrain;andthus,impacttheirpharmacologyeffects [8].Therefore,thestudyoftheirfunctionand expressioninthe endothelialand epithelialbarriersisnecessary tobetter under-standtheADMEofdrugsandtheirmetabolites.Thequantification ofABCtransportersattheBBBisgenerallyperformedonsamplesof micro-vesselsextractedusingaprotocolwelldescribedbyDauchy etal.[9].Itallowstoobtainasampleenrichedinendothelialcells butpollutedbydifferentcellsorcellularfractionssuchas peri-cytes,astrocytesandneurons[10].Thedetermination ofcertain markers,suchasATPase(a1,a2anda3)enablestoevaluatethe proportionofendothelialcellsinthesampleandbetterquantify the transporters[11]. MS-based absolute quantification of pro-teinsisusuallyperformedbyabottom-upapproach;whichimplies severalpreparationstepswherecaremustbetakenin orderto minimizevariabilityandensurereproducibility.Proteinextraction andfractionationisoftenfollowedbycleaningstepsbefore pro-teindenaturationandenzymatichydrolysis(mostfrequentlywith trypsin).Thesamplesarethenanalyzedbyliquidchromatography coupledtotandemmassspectrometry(LC–MS/MS)inmultiplexed selectedreactionmonitoring(SRM/MRM)toquantifyspecific tar-getpeptides[1,12]asasurrogateoftheprotein.Inordertoachieve anabsolutequantificationinnon-arbitraryunits(e.gpmol·mg−1_of proteins),stableisotopelabeled(SIL)internalstandardsareadded tothesampleafterdigestionintheformofsynthetichomologous peptides(AQUAstrategy[12]).

OneofthemajorissuesofMS-basedproteinquantificationisthe lackofuniversalsampletreatmentbecauseofthehighvarietyof

physicochemicalpropertiesofproteins.Inaddition,protocolsoften consistofmultiplesteps,whichincreasetheriskforinter-or intra-assayvariability.Therefore,itisimportanttocarefullyevaluateand validateallthestepsoftheanalysistoachievethedesiredaccuracy andprecision[13].

ThequantificationofmembraneproteinsasATP-binding cas-sette(ABC)transportersattheBBBandotherbarriersisalready describedintheliterature.TheTableS-1summarizesthemajor analyticalfeaturesoftheseveralavailablemethodsinthefieldon P-gpandBcrp.Kamiieetal.[1]developedanAQUAmethodfor thequantificationofmembranetransporterproteins,includingthe selectionofcandidatepeptideprobesandthesensitivityand accu-racyofthemulti-channelMRManalyses.Nevertheless,theydidnot performananalyticalvalidationofthedosagemethodaccordingto theFDAguidelines[13]asPrasadetal.[6]didforthe determina-tionofBCRPintheliverand,independently,forP-gpinthesame tissue[5].Groeretal.[7]andHarwoodetal.[14]alsovalidated analyticallythequantificationofPgpandBCRPinhumanintestine inaccordancewiththeseguidelines.However,theseauthorsdid notvalidateanymembranemarkerproteins(suchasATPAse).

EachstudydescribedinTableS-1usedonlyonepeptideper proteinforthequantificationanddidnottakeintoaccountthat thedigestionefficiencymayvarydependingonthepeptideas dis-cussedbyPrasadetal.[15],whichimpliesthatdigestionprotocol mustbeproven.Inthesestudies,theauthorsuseddigestion proto-colsalreadydescribedintheliterature[1,5,6,14,16],controlledthe digestionthroughtheCVvalueofaQC(<5%)[16]oroptimizedthe protocolbythemselves[7].Zhangetal.[17]suggestedcontrolling theP-gpdigestionthroughtheuseofasurrogatedigestionpeptide intheQCsamplesandHarwoodetal.[14]throughtheQconCAT technique.

Gröeretal.[7]andZhangetal.[17]weretheonlyauthors dis-cussingthematrixeffect.Thefirstdidnotstudyitbecausethey supposedthatmatrixeffectsareofminorimportancebecausethey usedalonggradientelutionandhigh-resolutionchromatography. Zhang et al. [17] discussedthe roleof the internalstandard in theminimizationofthepotentialinconsistencyintheionization bymassspectrometerofanalytesincomplexbiologicalmatrixes. However,mostoftheauthorsmadestandardsandQCsamplesina complexmatrix.Tominimizeapotentialmatrixeffectonthe quan-tification,thesesolutionsshouldbemadeinamatrixidenticalto that ofthesamplestobeassayed.Butthelatteraremembrane extractsthatusuallycomefromhumanoranimaltissuesorcells that are not alwaysavailable in largequantities. To mimic the matrix ofthesamplestheauthorsusedthebufferofthe mem-braneextractionkit[5,6],orsolutionsofpeptidesresultingfrom thedigestionofBSA[7,17]or,morespecificallyforQC, ¨true¨samples (membraneextractionofMDCKorHEK293cells)[16,17]forwhich they knewthe membrane protein concentration and that they spikedwithpeptidestobeassayedatknownconcentrations.None ofthepreviousworkscomparedthematrixeffectthatmayoccur onthedetectionofthepeptidescontainedinthesesolutions(more orlesscomplex)versusthesamepeptidesinaqueoussolution.

Therefore,themainobjectiveinthisworkistodevelopand vali-dateaLC–MS/MSmethodaccordingtotheFDAguideline(released

inMay2018[13])forabsolutequantificationofthemouseandrat membraneproteinsBcrpandP-gp,inadditiontothemembrane marker Na+_/K+ _{ATPase using isoform-specific peptides as} cell-markers.Themethodwasusedtodeterminetheexpressionofthese proteinsatthebraincorticalvesselsofratandmouse,andplasma membraneproteinsofmousekidneyandliver.Wehavestudied moreparticularlythreecriticalpointswhichcanmodifythequality ofthequantification.First,weverifiedthatthedigestion proto-colusedwasoptimizedfordigestionofmembraneproteinsafter precipitationwith methanol-chloroform-watersystem.We also studiedtheabsenceofimpactfromahighcomplexmatrixonthe quantificationperformance,thequantificationlinearity,accuracy, precision,LODandLOQdeterminations.Andfinally,westudiedthe peptidestabilityforthewholesampletreatment.Importantly,to improvethequantification,weemployedtwodifferentpeptides forBcrp,threeforP-gpandisoform-specificpeptidesforAtp1a,in additiontoapreviouslyusedmulti-isoformprobepeptide[1].

2. Materialandmethods 2.1. Chemicalsandreagents

Bovineserum albumin(BSA)and dextran(molecularweight 70.000)weresuppliedbySigmaAldrich(SaintQuentinFallavier, France). Protease Inhibitor Cocktail cOmplete Mini® _was pro-videdby Roche(Bâle, Switzerland).Sequencing grade Modified Trypsin,MS-graderLys-CandProteaseMAXsurfactantwere pur-chasedatPromega(Charbonnières-les-Bains).Someofthepeptides wereprovidedbyPepscan(Lelystad,TheNetherlands).AllFmoc protectedaminoacids,preloadedWangresinandpeptide synthe-sisreagentswereprovided byNovabiochem® _{(MerckMillipore,} Darmstadt,Germany).15_Nand13_{Clabeled(with98%ofisotopic} enrichment) Fmoc-protected amino acids came from Sigma-Aldrich.DMF(dimethylformamide)andpiperidineweresupplied byCarloErbaReagents(ValdeReuil,France).

2.2. Peptidesynthesis

The synthesis of peptides was performed in solid phase by using Fmoc chemistry and Fmoc-amino acid preloaded Wang resin (0.1mmol, 0.6mmol·g−1_{) on microwave assisted} CEM-Liberty1synthesizer,withDIC/Oxymapureascouplingreagents. Fmoc deprotectionwas achieved by20% (V/V) piperidine with 0.1mol·L−1 _{Oxymapure in DMF. After synthesis, peptides were} cleaved from resin witha simultaneous removal of side chain protectionsthroughatreatmentwitha10mLsolutionofTFA con-taining 2.5%water and 2.5%triisopropylsilane (V/V)(TIPS). The resinwasthenfilteredoffandthefiltratewasconcentrated, pre-cipitatedintocolddiethyl esterand collectedbycentrifugation. PeptideswerethenpurifiedonShimadzusemi-preparativeHPLC systembyusingaGRACEVydacProteinandPeptide218TPcolumn (10×250mm)andanalyzedonaShimadzuProminenceLC-20AD HPLCbyusingaGRACEVydacProteinandPeptide218TPcolumn (4.6×250mm),withina linearA–Bgradient(A:0.1% (V/V)TFA aqueous;B:0.09%(V/V)TFAin70%(V/V)acetonitrileaqueous)ata flowrateof2mLmin-1_{forpurificationand1mLmin}-1_foranalysis. Puritywasabove99%forallthepeptidessynthetized,exceptfor AAVPDAV[+6]GK(98.7%).Themolecularweightofpeptides was characterizedbyhighresolutionMALDI-TOFmassspectrometer. Thepeptideconcentrationinstandardsolutionswasdetermined byAminoAcidAnalysis(AAA)aftertotalacidhydrolysis.

2.3. Biologicalsamples

2.3.1. Yeastmicrosomalfraction

Yeastmicrosomalfractionwasobtainedbylysingthecellswith anUltraturrax®

(IKA®

-WerkeGmbH&Co.KG,Staufen,Germany) ina250mmol·L−1_{sucrosebuffer(with20mmol·L}−1_TrispH7.4and 5.4mmol·L−1_{EDTA).Afterclarifyingthesamplebycentrifugation} (15min.at10,000g,4◦_{C),themicrosomalfractionwaspelleted} by ultracentrifugation (1hat 100,000g, 4◦_{C) and recovered in} 250mmol·L−1_{sucrosebuffer.}

2.3.2. Animals

Male C57BL/6 (10–12 weeks old) mice and male Sprague–Dawleyrats(5–7weeksold),bothprovided byJanvier Labs (Le Genest-Saint-Isle, France) were handledin accordance withtheEuropeanCommunitiesCouncilDirective.Theanimals werehousedinastandard(non-enriched)environmentona 12/12-hlight-dark cyclein atemperature-controlled room(22±1◦_C). Foodandwaterwereprovidedadlibitum.Weendeavoredto mini-mizethenumberofanimalsusedandtheirdiscomfort.Theanimals wereanesthetized withKetamine/Xylasine, exsanguinated with physiologicalserumandeuthanizedbydecapitation.Themouse kidneysandbrainswereflashfrozeninliquidnitrogen,whilerat brainsweretreatedfreshly.

2.3.3. PlasmaMembraneProtein(PMP)fractionsofmousekidney andmouseliver

PlasmaMembraneProtein(PMP)fractionsofkidneyandliver wereobtainedaspreviouslydescribed[18]withminor modifica-tions,byusinganUltraturrax®_{forhomogenizationanddifferential} centrifugationwitha38%(W/V)sucrosecushionforPMP enrich-ment.

2.3.4. Mousebraincorticalvessels

Mouse brain cortical vessels were obtained as previously described[9,19]withsomemodifications,whilekeepingthe sam-plesat4◦_{C.Afterthawingthebrains,thecortexesweredissected} andcleanedofwhitematterandthemeninges.5braincortexes werepooledandchoppedinbuffer(HBSSand10mmol·L−1_HEPES). The suspension was centrifuged(5min. at 600g, 4◦_{C) and the} pelletwassuspendedinthesamebuffersupplementedbyan enzy-maticmixture.Thesampleswereincubatedat37◦_Candpelleted by centrifugation (15min. at 5000g, 4◦_{C). The pellet was} sus-pendedinsuspensionbuffer(HBSSwith10mmol·L-1_HEPES)with 17.5%(w/w)dextranandcentrifuged(30min.at4500g)to sep-aratemyelinandothercontaminantsfromthebrainvessels.The supernatantwaseliminated,andthepelletsuspendedin suspen-sionbufferwith1%(w/w)BSA,beforefiltrationbyusinga10␮m nylonmesh.Theretainedvesselswererecoveredinthesamebuffer andcentrifuged(5min.at600g,4◦_{C).Thepelletwassuspended} in suspensionbufferwithoutBSAand centrifugedagain(5min. at600g).Aftereliminatingthesupernatant,thevesselswere col-lectedinahypotonicbuffer(10mmol·L−1_{TrispH7.4,10mmol·L}−1 NaCl,1.5mmol·L−1_{MgCl2)supplementedwithproteaseinhibitor} cocktail,incubatedfor15minandsonicatedinBioruptor®_inhigh modefor5minwithon/offcyclesof30s.Sampleswerecentrifuged (10min.at10,000g,4◦_{C)andthesupernatantwasrecovered.} 2.3.5. Ratbraincorticalmicrovessels

Ratbraincorticalmicrovesselswereisolatedfromfreshtissueas previouslydescribed[19],byusingamechanicalhomogenization. Theprocedurewassimilartothemousevessels,butthe incuba-tionwithenzymeswasreplaced bymechanical disruption.The mincedsampleswerehomogenizedinaPotter–Thomas homog-enizer(KontesGlass,Vineland, NJ,USA) (0.25mmclearance)by using15to20up-and-downstrokesat400rpm.Microvesselswere

isolated by filteringthesamples thougha 100␮m nylonmesh followedbyasecondfilterof10␮m.Proteinswereextractedby suspendingthefinalpelletinthehypotonicbuffersupplemented withproteaseinhibitor cocktail,incubatedfor 15minand soni-catedinBioruptor®_{inhighmodefor5minwithon/offcyclesof} 30s.Sampleswerecentrifuged(10min.at10,000g,4◦_C)andthe supernatantwasrecovered.

2.4. Proteindigestion

ThetotalproteindeterminationwasperformedbyusingtheBCA ormicro-BCAProteinAssayKits,followingtherecommendations ofthesupplier(Thermo Scientific,Illkirch,France).Theproteins insamplesweredigestedinsolutionaspreviouslydescribed[1,2] withsomemodifications.Briefly,proteinsweredenaturedin dena-turing buffer (7mol·L−1 _{guanidine hydrochloride, 10mmol·L}−1 EDTA,500mmol·L−1_{TrispH8.5),reducedbyDTT(1.4mmol·L}−1₎ and alkylated by iodoacetamide (2.9mmol·L−1_{). Proteins were} then precipitated by using a methanol-chloroform-water sys-tem. The pellets wereresuspended in 6mol·L−1 _{ureaand 0.2%} (W/V)ProteaseMaxTM_{detergent.Aftera10-minuteincubationand} agitation at room temperature, thesamples were diluted with 0.1mol·L−1 _{Trisbuffer(pH 8.5)to afinal ureaconcentrationof} 1.4mol·L−1 _{and0.05%(W/V)ofProteaseMax}TM_{beforesonicating} foracompleteresuspension.rLysCendoproteasewasaddedtothe samplesinanenzyme-proteinmassratioof1:50anddigestedat roomtemperaturefor3h.Proteinswerethendigestedwithtrypsin (enzyme-proteinmassratio=1:100)byovernightincubation(16h) at37◦_{C.Thestableisotope-labeled(SIL)peptidemixturewasadded} beforestoppingthedigestionbyaddingformicacid.Thesamples weredriedinacentrifugalvacuumconcentrator(Maxi-DryLyo, HetoLabEquipment,Denmark),storedat−80◦_{Candsolubilized} justbeforeanalysis,byusingamixtureof10%(V/V)acetonitrile, 90%(v/v)waterplus0.1%(V/V)formicacid.

2.5. Analysisbyultrahighperformanceliquidchromatographyon linewithtandemmassspectrometry

2.5.1. UHPLCMS/MS

All the Mass Spectrometry analyses were performed on an ACQUITY UPLC H-Class®

System on line with a Waters Xevo®

TQ-Smassspectrometer(Waters,Manchester,UK).5␮Lofa solu-tionat1gL−1_{ofproteins(beforetrypsicdigestion)wereinjected} on thecolumn. Peptides were separated by using anACQUITY UPLCBEH®

C18column(PeptideBEH®

C18Column,300Å,1.7␮m, 2.1mmX100mm;Guyancourt,France)ina34mingradientgoing from100%aqueousmobilephase(waterand0.1%formicacid(V/V)) to35%oforganicmobilephase(ACNwith0.1%formicacid(V/V)) inaqueousmobilephase,ataflow-rateof0.5mL/min,at30◦_C.

ThemassspectrometerwasoperatedinMRMmodebyusing positiveelectrosprayionization(ESI)withionspraycapillary volt-age at 2.80kV. The resolution of the quadrupoles was 0.75Da (FWHM)aftercalibrationusingortho-phosphoricacid(0.1%)and resolution verification with Sodium Iodide (0.1mgmL−1_{) and} Cesium Iodide (2,5␮g mL−1) in positive mode (m/z 102.1300, 772.4610, 1372.0379 and 1971.6149), as recommended by the manufacturer (ref. 700005471, Waters, Manchester, UK). After manualoptimization,dryinggasflow-ratewassetto1000L/hwith atemperatureof650◦_{C.TheAQUAapproach[12]wasusedforthe} targetedquantificationofselectedpeptides.Methoddevelopment isdetailedinFig.S-1.Skyline[20]software(version3.1.0.7382) wasusedfortheMRMmethoddevelopment,includingthe colli-sionenergyoptimization,andthepeakintegration.MassLynxv4.1 (Waters,Manchester,UK)wasusedtopilotthemass spectrome-terandinitialinspectionofchromatograms.Collisionenergieswere optimizedbothmanuallyandwiththehelpofSkylineaspreviously

described[21].Briefly,formanualoptimizationastandardsolution ofeachpeptideat100pmol·mL−1_{wasanalyzedbydirectinfusion} whilechangingtheCEuntilhighestsignalintensitywasobtained.In addition,amixofstandardpeptidesat25pmol·mL−1_wasanalyzed usingaMRMmethodcreatedbySkylinewith5differentvaluesof CEincludingthepredictedoptimalCE,-6,-3,+3and+6eV.Results wereimportedtoSkylineandtheCEgivingthehighestpeakareafor mostofthetransitionsofapeptidewasselected;obtainingsimilar resultstomanualoptimization(Table1).

2.5.2. Datatreatment

AllthechromatogramswereevaluatedwithSkylinesoftware byusingtheinternalstandardmethodandpeak-arearatiofor cal-culation.QuaSAR[22],integratedasaplugininSkyline,wasused tocalculate thelimit ofdetection (LOD),limit ofquantification (LOQ)andlinearregressionequationsfromthecalibrationcurves foreachtransitionsurveyed(Table2).ThispluginusestheAuDIT [23]algorithmtoevaluatethepresenceofinterferencesby calcu-latinganadjustedp-valueoftheprobabilityofsignalinterferences foreachtransitionaccordingtotherelativeratiosbetweenlight andheavytransitions.Atransitionismarkedas“bad”whenthe combinedp-valueisinferiortoathreshold(10−5_)orthe%CVof thelight-to-heavyratiobetweentheinjectionreplicatesishigher thantheacceptedvalue(20%).Transitionsareconsidered“good” whennoneoftheseconditionsaresatisfied,makingthemsuitable forquantification.TheLODiscalculatedbyusingthelight-to-heavy ratioandtakesintoaccounttheestimatedaverageresponse (light-to-heavyratio)valuesofblanksamples,thestandarddeviationof blankandlowconcentrationpointsandthenumberofreplicates [23].LLOQwasthenestimatedas3xLOD.Thecalibrationequations areobtainedfromarobustlinearfitbyusingleastmedianofsquares regression.Thesecalibrationequationswereusedtocalculatethe LODandLOQvaluesinpmol·mL-1_units.

Thepeptideabundanceinsamplesandqualitycontrols(QCs) were calculated for each transition of the target peptides by using the calibration equations and home-developed R scripts and thentransformedintofmolper␮g oftotal protein.Finally, home-developedRscriptswereusedforallsubsequentstatistical evaluations.Reportedexpressionvaluescorrespondtothemean from3or4transitions;exceptforAAVPDAVGKwhichwas quan-tified byusing2 transitions.Thereported accuracy(DEV%)and precision(%CV)werecalculatedbyusingthepeptideabundance fromthetechnicalreplicatesof sampletreatmentforbiological samplesorpeptidemixinganddilutionforthecalibrationcurves andQCs;therefore,thevariabilitybetweenthetransitionsofasame peptideisnotconsidered.

2.6. Developmentoftheabsoluteproteinquantification(AQUA) method

2.6.1. PeptideselectionforAQUAmethod

Theabsoluteproteinquantification(AQUA)[12]ofanalyte pro-teins(Bcrp,P-gpandNa+_/K+_{ATPase)wasperformedbycombining} aninsilicopeptideselectionwithanexperimentalvalidation(Fig. S-1).Briefly,thepossibleproteotypicpeptideswereselectedin sil-icobyusingcriteriasuggestedpreviously[1,24](TableS-2),with the help of bioinformatics tools (Table S-3). The Protein Infor-mationResource(PIR)peptidesearch(http://pir.georgetown.edu/ )[25]wasparticularlyusefultoverifypeptidespecificityasit per-formsaquicksearchforthesequencesversustheUniProtKBentries. The peptides selectedare specifictothecorresponding protein homologuesinmouse,ratandhumanproteome;exceptfor pep-tideVGTQFIRfromBcrp,whichisnotpresentintheratprotein. Finally, thetarget peptides for thecontrol proteinand interest proteinswerechosenbyanalyzingproteindigestsbyLC–MS/MS whichusesanunscheduledMRMmethod.Atleast2peptideswere

Table1

Targetpeptides,MRMparametersusedintheUHPLC-MS/MSanalysis.

Protein Peptide Light Heavy FragmentIon CV(V) CE(eV)

N◦ _{Sequence(light/heavy) Precursorm/z Productm/z Precursorm/z Product}

P-gp(a) 1 NTTGALTTR/NTTGALTTR[+10] 467.8 719.4 472.8 729.4 y7 35 16 618.4 628.4 y6 561.3 571.3 y5 490.3 500.3 y4 2 LANDAAQVK/LANDAAQV[+6]K 465.3 816.4 468.3 822.4 y8 35 16 745.4 751.4 y7 631.3 637.4 y6* 516.3 522.3 y5 P-gp(a/b) 3 IATEAIENFR/IATEA[+4]IENFR 582.3 979.5 584.3 983.5 y8 35 21 749.4 753.4 y6 678.4 678.4 y5 Bcrp 1 SSLLDVLAAR/SSLLDVLA[+4]AR 522.8 757.5 524.8 761.5 y7 35 18 644.4 648.4 y6 529.3 533.4 y5 430.3 434.3 y4 2 VGTQFIR/VGTQFIR[+10] 410.7 721.4 415.7 731.4 y6 35 14 664.4 674.4 y5 563.3 573.3 y4 435.3 445.3 y3

Na+/K+ATPaseAtp1a1/2/3 AAVPDAVGK/AAVPDAV[+6]GK 414.2 756.4 417.2 762.4 y8* 35 14

685.4 691.4 y7

586.3 592.3 y6

489.3 495.3 y5*

Na+/K+ATPaseAtp1a1 IVEIPFNSTNK/IVEIPFNSTNK[+8] 631.3 1049.5 635.4 1057.5 y9 35 22

920.5 928.5 y8

807.4 815.4 y7

710.3 718.4 y6*

Na+/K+ATPaseAtp1a2 GIVIATGDR/GIVIATGDR[+10] 451.3 731.4 456.3 741.4 y7 35 16

632.3 642.3 y6

519.3 529.3 y5

448.2 458.2 y4

Na+/K+ATPaseAtp1a3 GVVVATGDR/GVVVA[+4]TGDR 437.2 717.4 439.2 721.4 y7 35 15

618.3 622.3 y6

519.3 523.3 y5

448.2 448.2 y4

selectedandusedforthequantificationofeachproteinincluding

somesequencespreviouslyusedfortheirquantification[1,5,6]and

somenovelsequences(Table1). 2.6.2. Proteindigestionoptimization

Theproteindigestionefficiencywasinvestigatedbyusing kid-neytissue(availableinhigherquantitythanmicrovessels)which wasdigestedfor4h(protocolA),16h(protocolB)and24h (proto-colC)at37◦_{CwiththereagentquantitiesdescribedinSection2.5;} for16hat37◦_{CwithoutrLysCandtrypsinwithenzyme-protein} massratio=1:100(protocolD),for 16hat37◦_{CwithrLysCand} trypsinatverylowconcentration(1:1000insteadof1:100) (proto-colE)orahigherconcentration(1:50insteadof1:100)(protocolF). Proteinlinearizationbyheatingat95◦_{Cbeforethereductionstep} androomtemperature(25◦_{C)trypsindigestionwerealsotested} (protocolGandH,respectively).Ineachcase50␮gofproteinswere digestedintriplicateandthepeptideabundanceweredetermined forP-gp(a),P-gp(a/b),BcrpandNa+_/K+_ATPases.

2.7. Validationoftheabsoluteproteinquantification(AQUA) method

Themethodvalidationwasperformedbasedonthe recommen-dationsoftheFoodandDrugsAdministrationBioanalyticalMethod ValidationGuidanceforIndustry[13].

2.7.1. Preparationofcalibrationcurvesandqualitycontrols(QCs) Calibrationcurvesinpure solutionwereprepared inthree dilution replicates at three different days with three different batchesof peptides in a mixtureof 10%(V/V) acetonitrile,90% (V/V) water plus 0.1% (V/V) formic acidby successive dilution

beforeaddinga constantamountofstableisotope-labeled pep-tides. Lightpeptideconcentrations covered a range from 0.125 to 125pmol·mL−1 _{(0.125, 0.25, 1.25, 2.5, 12.5, 25, 62.5 and} 125pmol·mL−1_{).TwoextrapointswereincludedfortheNa}+_/K+ ATPasepeptides(300and510pmol·mL−1_{).Calibrationcurveina} complexmatrixwaspreparedasasurrogateofthesampleblank matrix.Thecalibrationpointswerepreparedin triplicate,using the samethree batches of stock standard peptidemixture and internalstandardsasinthepuresolutionbutinacomplex back-groundmatrixconsistingofadigestofmicrosomal-yeastproteins (S.cerevisiae)ataconcentrationof1␮gofhydrolysateper␮L (mim-ickingtheconcentrationofsamples).Thefinalconcentrationswere calculated tocover a rangefrom0.195to25pmol·mL−1 _(0.195, 0.391,0.781,1.563,3.125,6.25,12.5,25pmol·mL−1_).QCsamples werepreparedbyusingadigestofyeastmicrosomalproteins,as explainedabove,infourconcentrations:neartoLLOQ(belowthree timestheLLOQ,seeTables3andS-4),low,mediumandhigh(10, 20and60pmol·mL−1_{,respectively).Threebatcheswereprepared} indifferentdays.

2.7.2. Peptidepurity,standardselectivity

Purityandconcentrationofreferencestandardswasassessed forallthepeptidessynthetizedinourlaboratoryasmentionedin section2.1.

Cross-interferencebetweenlabeledandunlabeledstandards wastestedbyanalyzingzerocalibrators;meaningthemixof heavy-labeledstandardpeptideswithoutlightpeptides,bothinpureand complexmatrix.

Table2

Targetpeptides,MRMparametersusedintheUHPLC-MS/MSanalysis,calibrationequations,R2_{andmatrixeffectcomparisonofslopes.}

Protein:PeptideSequence(light/heavy) FragmentIon CalibrationEquation SlopeMatrixeffect

LowComplexity Highcomplexity

Slope Yintercept R2 _{Slope Yintercept R}2

P-gp(a): NTTGALTTR/NTTGALTTR[+10] y7 0.0612 0.0008 0.9983 0.0587 0.0303 0.9973 −4.0% y6 0.0622 −0.0001 0.9988 0.0581 0.0001 0.9965 −6.5% y5 0.0618 0.0006 0.9990 0.0593 −0.0044 0.9916 −4.0% y4 0.0625 −0.0031 0.9995 0.0584 −0.0004 0.9969 −6.5% P-gp(a): LANDAAQVK/LANDAAQV[+6]K y8 0.0600 0.0014 0.9759 0.0578 −0.0014 0.9968 −3.6% y7 0.0574 0.0023 0.9931 0.0553 0.0008 0.9954 −3.5% y6* 0.0596 −0.0001 0.9968 0.0581 0.0043 0.9944 −2.5% y5 0.0586 0.0004 0.9939 0.0570 0.0018 0.9970 −2.8% P-gp(a/b): IATEAIENFR/IATEA[+4]IENFR y8 0.0594 0.0014 0.9994 0.0590 0.0013 0.9978 −0.8% y6 0.0614 0.0021 0.9992 0.0615 0.0016 0.9979 0.1% y5 0.0607 0.0024 0.9992 0.0589 0.0032 0.9972 −3.0% Bcrp: SSLLDVLAAR/SSLLDVLA[+4]AR y7 0.0709 0.0030 0.9982 0.0687 0.0040 0.9976 −3.1% y6 0.0710 0.0026 0.9985 0.0687 0.0014 0.9968 −3.3% y5 0.0713 0.0027 0.9989 0.0692 0.0019 0.9961 −3.0% y4 0.0709 0.0042 0.9989 0.0701 0.0024 0.9984 −1.0% Bcrp: VGTQFIR/VGTQFIR[+10] y6 0.0597 0.0008 0.9991 0.0540 0.0020 0.9969 −9.5% y5 0.0598 0.0003 0.9988 0.0562 0.0009 0.9951 −6.2% y4 0.0589 0.0021 0.9992 0.0560 0.0011 0.9973 −5.0% y3 0.0589 0.0006 0.9989 0.0564 0.0001 0.9972 −4.3% Atp1a1/2/3: AAVPDAVGK/AAVPDAV[+6]GK y8* 0.1100 −0.0066 0.9934 0.1383 −0.4152 0.9691 25.7% y7 0.1099 0.0026 0.9990 0.1097 −0.0026 0.9938 −0.2% y6 0.1098 0.0049 0.9989 0.1097 0.0012 0.9962 −0.1% y5* 0.1097 −0.0011 0.9890 0.1004 0.1065 0.9810 −8.5% Atp1a1: IVEIPFNSTNK/IVEIPFNSTNK[+8] y9 0.0555 0.0022 0.9948 0.0532 −0.0005 0.9982 −4.0% y8 0.0565 0.0031 0.9940 0.0539 0.0002 0.9957 −4.6% y7 0.0568 0.0019 0.9971 0.0530 0.0000 0.9977 −6.7% y6* 0.0541 0.0040 0.9863 0.0535 −0.0026 0.9908 −1.0% Atp1a2: GIVIATGDR/GIVIATGDR[+10] y7 0.0770 0.0009 0.9996 0.0723 −0.0001 0.9964 −6.1% y6 0.0796 0.0007 0.9997 0.0739 −0.0012 0.9986 −7.2% y5 0.0800 0.0008 0.9996 0.0745 −0.0022 0.9974 −6.8% y4 0.0776 0.0019 0.9991 0.0757 0.0003 0.9969 −2.4% Atp1a3: GVVVATGDR/GVVVA[+4]TGDR y7 0.0606 0.0025 0.9987 0.0584 0.0037 0.9956 −3.6% y6 0.0617 0.0028 0.9981 0.0611 −0.0001 0.9986 −1.0% y5 0.0633 0.0028 0.9986 0.0605 0.0028 0.9971 −4.3% y4 0.0600 0.0025 0.9942 0.0593 0.0048 0.9953 −1.2%

CalibrationequationsandR2_{wereobtainedfromtheanalysisofacalibrationcurvepreparedintriplicate(eachreplicatewasinjectedonce).Allthepeptidesarecommon} betweenthemouseandratproteins,exceptforVGTQFIRwhichisnotpresentinratBcrp.Heavyisotopelabeledresiduesarefollowedbytheirmassshiftinbrackets(rounded tothenearestinteger;e.g.[+10])betweenbrackets(e.g.K[+8]).CV=ConeVoltage;CE=CollisionEnergy;IsotopeType:L=Light,H=Heavy.Thematrixeffect(ME)onslope wascalculatedusingthefollowingequation:ME=(SHC-SLC)/SLC;wereSHC=slopeinhighcomplexitymatrixandSLC=slopeinlowcomplexitymatrix.*:theseionswere notusedforquantificationduetopossibleinterferences.

Table3

Lowerlimitofdetection(LOD)andlowerlimitofquantification(LOQ)asdeterminedinlowandhighcomplexitymatrices.ValuesareinpmolmL−1_.

Protein Peptide Conc.inLLOQQCs Lowcomplexitymatrix Highcomplexitymatrix RatioLOQHigh/Low

N◦ _{Sequence LOD LOQ LOD LOQ}

P-gp(a) p1 NTTGALTTR 3 0.125 0.275 0.205 1.65 6.0 P-gp(a) p2 LANDAAQVK 2 0.085 0.334 0.292 1.02 3.1 P-gp(a/b) p3 IATEAIENFR 2 0.053 0.207 0.214 0.752 3.6 Bcrp p1 SSLLDVLAAR 1.5 0.117 0.468 0.227 0.751 1.6 Bcrp p2 VGTQFIR 1 0.086 0.285 0.164 0.567 2.0 Na+_/K+_ATPase Atp1a1/2/3 AAVPDAVGK 8 1.07 3.31 0.318 0.976 0.3 Atp1a1 IVEIPFNSTNK 1.5 0.598 1.86 0.270 0.817 0.4 Atp1a2 GIVIATGDR 1.5 0.047 0.158 0.261 0.750 4.8 Atp1a3 GVVVATGDR 2 0.078 0.316 0.290 0.961 3.0

2.7.3. Calibrationcurvelinearity,limitofdetection(LOD)and

limitofquantification(LOQ)

Thelinear equation,limit of detection(LOD)and limitof

quantification (LOQ) wereobtainedfrom theanalysisof three

batchesofcalibrationcurvesinpuresolutionandcomplexmatrix

analyzedseparately(singleinjection).Thevalueswereobtained

usingQuaSAR[29]asexplainedinsection2.5.

2.7.4. Accuracyandprecision

Accuracyis expressedasthedeviationtothenominalvalue (%DEV)andprecisionasthepercentofcoefficientofvariation(%CV).

Accuracyandprecisionofthecalibrationcurveswereevaluated bothinpureandcomplexmatrices.Within-runaccuracyand pre-cisionwerecalculatedbyanalyzingintriplicatethefourlevelsof QCsincomplexmatrixandonebatchofcalibrationcurvepointsin puresolution.Betweenrunaccuracyandprecisionwasassessed byanalyzingthethreebatchesoflow,mediumandhighQCsamples onthreedifferentdays.

2.7.5. Matrixeffect

Theabsenceofimpactfromthecomplexmatrixonthe quan-tification performance was evaluated by comparing the slope,

Fig.1.Comparisonofdigestionprotocolsperpeptide.Measuredexpressionvaluesweredividedbythecontrolprotocol(standardprotocol)with16hofdigestion)toobtain arelativedigestionefficiency.Thegraydashed-lineindicatesanefficiencyof1(thestandardprotocol).BLQ=belowthelimitofquantification.Significantdifferencesmarked byasterisksforp-valuelowerthan0.05(*),0.01(**)and0.001(***)werestudiedusinglog10abundancevaluestofittoanormaldistribution(t-testwithBonferroni’s correction).

accuracyandprecisionbetweenthepureandcomplexmatrices. Thevaluesfromtheyeasthydrolysateshouldbetterrepresentthe limitsoftheanalyticalmethodinthebiologicalsamples.

2.7.6. Peptidestability

Freeze-thawstabilitywastestedbyusingthestocksolution containingthemixofstandardpeptides.Individualpeptidestock solutionsareallstoredat−80◦_{Cin100␮Laliquot.When} calibra-tionrangewasprepared,thesesolutionswerethawedandmixedto obtainastocksolutioncontainingeachpeptideattheconcentration of250pmol·mL−1_{.Thisstocksolutioncouldberefrozenandthawed} forfurtherhandling.Therefore,itsstabilitywasevaluatedafter1, 2,4and6cyclesoffreezingandthawingwith3daysintervals.

The stability of thepeptides after several cyclesof thawing thesampleshasnotbeentestedbecause,inthisstudy,wenever reinjecteda sampleafterjustoneorseveralfreeze/thawcycles. Similarly,wheninternalstandardsorcalibratorsareprepared,they arealiquotedinsingleusevolumes.

Sampleprocessing(digestion)stabilitywasevaluatedby spik-ing the yeast microsome fraction with light standard peptides eitherbeforeorafterovernightincubation withtrypsin(16hat 37◦_{C)duringthe proteindigestionprocedure. The SILpeptides} wereaddedaftertheincubationandsamplesweredriedand ana-lyzedasexplainedabove.

Autosamplerstabilityduringtheanalysis(i.e.at4◦_C)was eval-uated for3 typesofsamples: calibrationcurve points,QCsand mousekidneyplasmamembraneproteins.Twodifferentbatchesof calibrationcurveswereinjectedtwicewithanintervalof approx-imately30hbetweenthefirstandthesecondanalysis.EachQC(3

levelsfrom3differentbatches)orproteindigestsample(in tripli-cateofdigestion)wasinjectedthreetimes:atthebeginningofthe analysisseries,45hlaterand75hlater.

3. Resultsanddiscussion 3.1. Methoddevelopment

3.1.1. PeptideselectionforAQUAmethodandCEoptimization Theselection ofpeptidesfor proteinquantificationwas per-formedinsilicofollowingcriteriapreviouslysuggestedbyKamiie et al. [1] and enriched with suggestions from Ludwig and Aeberesold[24](TableS-2).Theuseofseveralbioinformatictools (Table S-3)allowedustoselectthesurrogatepeptides.ThePIR PeptideSearchwasparticularlyusefulfortheselectionof protein-specificpeptidesthatarepresentinthehomologuesofP-gp,Bcrp andtheNa+/K+ATPasepump(Atp1a1,2and3)frommouse,ratand humanandotherspeciesofinterestforpre-clinicalanalysis(Table S-5).Skyline[20]wasusedforchromatograminspectionanddata treatment.Thisstate-of-the-artsoftwarefacilitatedthe develop-ment,validationandanalysisoftargetedproteomicsexperiments. Collisionenergywasoptimizedbothmanuallyandwiththehelp ofSkyline[21]asexplainedinsection2.6.1.TheoptimalCE corre-spondedtotheonepredictedbythesoftwareor3eVbelowforall thepeptidesasshowninFig.S-2.

3.1.2. Proteindigestionoptimization

Fig. 1 shows the relative digestion efficiency calculated by normalizingagainstthestandardprotocolwith16hofdigestion

(protocolB).Atp1a2andAtp1a3peptidesarenotincludedbecause theseprotein isoformsarenot expressedinmouse kidney[26] and thus werenot detected. Significantdifferences were stud-iedusinglog10abundancevaluestofittoanormaldistribution (t-testwithBonferroni’scorrection).Threereplicatesofeach pro-tocol wereevaluated andthus someapparentdifferences were notstatisticallysignificant;nevertheless,thisexperimentallowed thedetectionofcriticalparametersonthesampletreatment. P-gppeptideNTTGALTTRpresentsahighLLOQandwasBLQinall thesamples.P-gppeptidesLANDAAQVKandIATEAIENFRwerenot detectedwhenLysCwasnotused(protocolD),whentrypsinwas usedatverylowconcentration(1:1000;protocolE),aftersample heatingat95◦_{Cfor5min(protocolG)orroomtemperature(25}◦_C) digestion(protocolH).Thedifferencesindigestionefficiencywere significantforAAVPDAVG(Atp1a1,2,3)andIVEIPFNSTNK(Atp1a1) byusinglowconcentrationoftrypsin(protocolE)orheatingthe proteins (protocolG), and for VGTQFIR (Bcrp) in this last pro-tocol. There were nosignificant differences in thedigestion of all the proteins by using protocols A, B and C with LysC and trypsin(enzyme-proteinmassratio=1:100;416and24h diges-tion,respectively);neitherwithprotocolFwheretrypsin-protein ratiowasof1:50.Althoughatrypsin-to-proteinratioof1:50to 1:20isoftenusedinproteindigestion,ourresultsindicatedthat the1:100ratioisenoughforcompleteproteindigestionafter pre-digestionwithLysCinaccordancetopreviouslyreportedprotocols fortargetedproteomicsofABCtransporters[1]andotherproteins [27].TherecoveryofP-gppeptideswasespeciallysensibletoall theconditionspossiblyduetoitslowabundanceinmousekidney. Inaddition,ABCtransportersandothermembraneproteinssuch astheNa+_/K+_{ATpasepumpareheat-sensibleascanbeobserved} intheresultsfromprotocolG(speciallyforP-gp).Someprotocols usea95◦_{Cincubationtohelplinearizetheproteinsfordigestion,} butthisshouldbeavoidedwhenquantifyingmembraneproteins. Otherprotocolsperformthetrypsindigestionunderroom tem-peraturetoavoiddegradation,butthishasproveninsufficientfor P-gpenzymatichydrolysis.Consequently,LysCandtrypsinwith enzyme-proteinmassratio=1:100areadequateandnecessaryto obtainacompletedigestionofproteinsat37◦_Cfor16h.

3.2. Methodvalidation

Severalnationalorinternationalagencieshaveprovided reg-ulatory guides for the validation of LC–MS based methods for thequantificationofdrugsandmetabolitesinpharmacokinetics studies,buttheydonot directlydeal withthequantificationof endogenousproteinsorbiomarkers.Onlytherecentlypublished BioanalyticalMethodValidationGuidancefor Industryfromthe AmericanFoodandDrugsAdministration(FDA)[13]mentionthat itcanbeextendedtotheevaluationofproteinbiomarkerlevels inbiologicalmatrices.Therefore,weusedthisguidefortheAQUA methodvalidation.

TheFDA guidestatesthat thecalibrationrange and theQCs shouldbemadeinthesamematrixasthesamples.However,we donothaveaproteinsamplefrommicro-vesselsthatdoesnot con-taintheproteinstobeassayed.Someauthorshavesubstitutedthe matrixbydifferentsolutions.Jietal.[28]usedthemembrane pro-teinfractionofwild-typeHEKcellsastheblankmatrixtoprepare standard solutionand themembranefractionof controlmouse livertissuefortheQCsamples;Zhangetal[23]andGröeretal. [8],solutionsofpeptidesresultingfromthedigestionofBSAfor standardandQC.ThepreparationofstandardsolutionandQCina hydrolysateofbrainmicro-vesselsproteinsisimpossiblefor ethi-calreasonsbecausemanyanimalswouldhavetobesacrificedfor thematrixpreparation.Inaddition,thesamplewithouttheanalyte proteinsisnotavailable.Therefore,wetestedthelinearity, accu-racy,precisionandstabilityforalltargetpeptidesnotonlyinthe

lowcomplexitysolution(calibrationcurvespreparedin10%(V/V) acetonitrile,90%(V/V)water+0.1%(V/V)FormicAcid)butalsoin yeastmicrosomalproteinstotesttheeffectofacomplexmatrix inthequalityofthequantitativemethod.Weusedyeast microso-malproteinsbecauseitcontainsahighvarietyofproteinsandits complexitycanbecomparedtooursamplesofinterest.Moreover, yeastproteinscanbeobtainedatacheappriceandthedigestionof theseproteinsdoesnotproduceanypeptidesredundantwithour targetpeptides.

3.2.1. Peptidepurity,standardselectivity

Thepurityofallthepeptidesusedwasabove99%,exceptfor AAVPDAV[+6]GK(98.7%)(Fig.S-3).Theconcentrationofthestock solutionswasdeterminedbyAAA(TableS-6).Theisotope enrich-mentofheavylabeledstandardsusedforthesynthesiswasabove 98%tominimizecross-contaminationtounlabeledpeptides.

Thereislessthan1%ofinterference(ratioof0.01)foralmost allthepeptidesbothinthepureandcomplexmatrices,although higherinterferencesareobservedinthesamplespreparedonYeast hydrolysate.PeptidesAAVPDAV[+6]GK(ATP1a1/2/3)and IVEIPFN-STNK[+8] (Atp1a1) showslightly higher interferences, but they are still below the FDA acceptance criteria of 5%. Thus, cross-interference between labeled and unlabeled peptides (isotopic effects)arenegligible.Inaddition,thesignalsatthelight transi-tionsforpeptideswitham/zdifference>2donotco-elutewith theheavy-standardsignal(Figs.S-4andS-5)anddonothavethe samerelativeintensity;thus,thesignalobservedcouldbedueto backgroundnoiseinsteadofcross-interference.

3.2.2. Calibrationcurvelinearity

Eachpeptidewasquantifiedbysurveyingfourdifferent frag-mentsfromthe[M+2H]++_{precursorasMRMtransitions(Table1).} Sometransitionsturnedouttobenoisyandwerethenusedonly forpeptideidentificationregardingtheircoelutionwiththeother transitions.Nevertheless,threetofourtransitionswereusedfor thequantificationofeachpeptide,exceptforAtp1a1/2/3 (AAVP-DAVGK)withonlytwo.Inordertoimprovethequantification,the averagepeptideabundanceswereobtainedthroughtheaverage valuesobtainedforitstransitions,whichhada%CVlowerthan15 inmostcases.

AlmostallthetransitionsevaluatedgaveR2_{valuesabove0.99} bothinthepureandcomplexmatrices(Table2), provinga cor-rectlinearfit;therebyshowingthattargetpeptidesaresuitable forquantification.Fig.2bshowsthechromatogramobtainedafter injectionofstandardsolutionsat12.5pmol·mL−1 _inamixtureof 10%(v/v)acetonitrile,90%(v/v)waterplus0.1%(v/v)formicacid (Fig.2aandb)andinyeastproteindigest(Fig.2eandf).

3.2.3. Limitofdetection(LOD),limitofquantification(LOQ) Limitsofdetection(LOD)andquantification(LOQ)foreach tran-sition(TableS-4)wereobtainedfromcalibrationcurvesprepared eitherinpuresolutionorinyeastmicrosomalproteindigest.The highestvalueswereconsideredastheLODandLOQatthepeptide level,whicharepresentedinTable3.Itiswellknownthatcoeluting substancescanhaveamajoreffectintheionizationanddetectionof targetpeptides;therefore,itisnotsurprisingthatthequantification limitsarehigherintheyeastdigestmatrixthanthepuresolution formostofthepeptides.Furthermore,thedifferencebetweenthe highandlow complexitymatrixwasdifferentfor eachpeptide, fromaratioof0.3(AAVPDAVGK)to6(NTTGALTTR,P-gp(a)).This highlightsthateachpeptidecanbesubmittedtodifferentmatrix factorsaccordingtoitsretentiontime.Therefore,careshouldbe takenwheninterpretinglimitsofquantificationfromcalibration curvespreparedinpuresolution.Thus,theLOQvaluesobtained fromtheyeastdigestwereusedinotherexperimentsinorderto consideranestimationofthebackgroundnoiseofacomplex

bio-Fig.2.ChromatogramsobtainedfromtheMRManalysisofstandardsolutionsinlowcomplexitymatrix(left:a,b,c,d)andcomplexmatrix(right:e,f,g,h).Fromtopto bottomareshown:Internalstandardheavypeptidesexample(a,e),unlabeledstandardscalibratorsatconcentration12.5pmol·mL−1(b,f),nearLLOQ(c,g)andnearLOD(d, h).

logicalsample.Fig.2showsexamplesofchromatogramsfromthe injectionofcalibrationpointsneartotheLLOQandLODofmostof thepeptidesfromcurvespreparedinamixtureof10%(V/V) ace-tonitrile,90%(V/V)waterplus0.1%(V/V)formicacid(Fig.2candd) orinyeastproteindigest(Fig.2gandh).Thisstudyhighlightsthe impactofmatrixonthebackgroundnoise,muchhigherinFig.2g andhthaninFig.2candd.

3.2.4. Accuracyandprecision

AccordingtotheFDAguidanceforbioanalyticalmethod vali-dation,thecalculatedconcentrationofQCsandcalibrationpoints ofbioanalyticalmethodshouldhaveaprecisioninferiorto15%CV andaccuracyshouldbewithin15%ofthenominalconcentration, exceptfortheLLOQpointswherethethresholdissetto20%.At least75%ofthecalibratorpointsand67%oftheQCsshouldsatisfy thesecriteriaforalltheanalytes[13].

Inadditiontothecalibrationcurves,fourlevelsofQCswere ana-lyzedasproposedbytheFDAguidance.TheQCsclosetoLLOQwere preparedwithspecificconcentrationsofeachpeptide(seeTable3) within3-foldtheLLOQinyeast;exceptfortheAtp1a1/2/3 surro-gate.ThispeptidehasaconsiderablyhigherLLOQinpuresolution andtheconcentrationwassetto8becauseitisexpectedinhigh abundancelevelsinoursamples.

Accuracy and precision of the calibration curves were evaluatedbothinpureandcomplexmatrices.Theestimated con-centration inboth matrices wascalculated using theequations obtainedfromthepurecalibrationcurve(Fig.3aandb),butthe valuesfortheyeastcalibratorswerealsoobtainedusingtheirown equations(Fig.3c)tocomparethevaluesasanothermeasureofthe matrixeffect(Section3.2.4).Inouranalysis,allcalibrationpoints abovetheLLOQpresentedanerrorandprecisionbelowthe thresh-oldof15%,bothinthepure(Fig.3a)andcomplex(Fig.3c)matrices forallthetargetpeptides.

Within-runaccuracyandprecisionwereevaluatedusingQCs fromyeasthydrolysate.TheQualityControlsamples(QCs)(Fig.4a) alsopresentedaverylow%CV,below5%forallthepeptidesexcept for theAtp1a3 surrogateathighconcentration levels,but they

wereallbelow15%.Nevertheless,ahigheraccuracydeviationwas generallyobserved;probablyasaresultofthematrixeffecton ion-izationorinterferences(i.e.coelutingmolecules).Almostallthe peptidespresentedadeviationbelowthethresholdof15%andonly theLowQCsofLANDAAQVK(P-gp)andAAVPDAVGK(Atp1a1/2/3) werebetween15and25%.Theseresultsconfirmthatthis analyti-calmethodallowspeptidequantificationwithasatisfyingaccuracy andprecision.Inaddition,as3differentreplicatesofpeptidemix anddilutionwereused,thisindicatesthattherewasalow inter-batchvariability.

Between-runsaccuracyandprecisionwerecalculatedfrom theanalysisthreedifferentdaysofthreebatchesoflow,medium andhighQCsamples.Weobserveda%CVand%Deviationbelow 15%forallthepeptides(Fig.4b).

3.2.5. Matrixeffect

Itiswellknownthatotheranalytesinthesamplecaninterfere withthequantificationintheanalytesduetodifferentfactorssuch asionizationcompetitionorinterference,whicharesummarized asthematrixeffect.TheFDAguidancementionsthatthereshould notbeamatrixeffectinthequantificationofbiomarkers,butdonot giveclearinstructionstomeasurethis[13].Consideringthatitis notpossibletoobtainenoughmicro-vesselssamplestostudythis, weusedyeastproteinhydrolysatetomimicthematrix.The cali-brationpointswerepreparedusingthesamethreebatchesofstock standardpeptidemixtureandinternalstandardstoavoidbiasdue topreparation.AsdetailedinSection3.2.3,wegenerallyobserved ahigherLOQinthecomplexmatrix.Inaddition,wecomparedthe impactonthecalibrationequationslope(Table2)andobserveda generaldecreaseinthecomplexmatrixslope,goingfrom0to-5% in70%ofthetransitions.Similarly,thedeviationofthe concentra-tioninthecomplexmatrixcalibratorsestimatedwiththeequation frompurecalibrators(Fig.3b)waswiderthanwhentheequation fromthesamematrixwasused,althoughstillwithinthelimitsof 15%.Theprecisionvaluesaremoreindependentfromthe equa-tionusedandthevaluesinFig.3bandcaresimilarandbelow15% forthenonLLOQcalibrators.AlthoughtheFDAguidancedoesnot

Fig.3. Precision(%CV)andaccuracy(%Deviation)forcalibrators.Calibrationcurveswerepreparedinlowcomplexitymatrix(a)(90%water,10%ACN+0.1%formicacid)and highcomplexitymatrix(b,c)(yeastdigest).Concentrationvalueswereestimatedusingtheequationsfromeitherthelowcomplexitymatrix(a,b)orthehighcomplexity (c).

Fig.4. Precision(%CV)andaccuracy(%Deviation)forQCs.Threebatchesofqualitycontrol(QC)sampleswerepreparedinhighcomplexitymatrix(yeastdigest)infour concentrations:neartoLLOQ(below3-fold),10,20and60pmol·mL−1_{.ThefourQClevelswereanalyzedthreetimesthesamedayforwithin-runevaluation(a)andinthree}

separatedaysforbetween-runevaluation(b)(LLOQ-nearwasnotincluded).

establishatolerancethresholdformatrixeffect,weconsiderthat valuesbelow15%andtheconservationoftheaccuracyalsobelow thisvalueshouldminimizetheimpactonthequantification. There-fore,weusedthecalibrationcurvepreparedinpuresolutionfor quantificationofsamples,tomakethemethodsimpler.

3.2.6. Peptidestability

Freeze-thawstabilitywasprovenforallthepeptides,asthey presentedarecoveryof98.5–100%aftersixfreezing/thawingcycles asshowninTableS-7.

Peptidestabilityduringdigestionwasstudiedinyeast pro-teinsamplesspikedwiththestandardlightpeptidesbeforeorafter thedigestion(TableS-8).Mostofthepeptidesusedinthepresent

studydidnotshowasignificantlydifferentpeptideamount(t-test onlog10abundancevalues,p-value>0.05).OnlyVGTQFIR(Bcrp) presented a significantly lowerconcentration (p-value=0.0109) whenthepeptidesfollowedthedigestionincubation, represent-ingaslightdegradation(-9%).Nevertheless,thesedifferencesare withintheacceptederrormargins(<20%)[13].Theseresults sug-gestthatthetargetpeptidesselectedaresufficientlystableinthe trypsinationconditionsused(16hat37◦_{C),indicatingthusthatthis} quantificationshouldnotpresentbiasduetopeptidedegradation atthisstep.

Stabilityinautosampler(4◦_{C)wasevaluatedbyreinjectingall} thecalibrationpoints30hafterthefirstanalysisandtheQC lev-elsafter45and75h(TableS9withoutobservinganydifference

Fig.5.ChromatogramsobtainedfromtheMRMofratbrainmicrovessels(a)andmousemicrovessels(b).

Table4

TargetedabsolutequantificationbyLC–MS/MS(MRM)ofthesurrogatepeptidesfromtheendogenousproteinsBcrp,PgpandNa+/K+ATPaseinmouseandrattissues.

Protein Peptide Proteinamountperpeptide(fmol␮g−1±s.d.)

N◦ _{Sequence Mouse-kidney} (PMP) MouseLiver (PMP) MouseCortical Vessels(WL) RatCortical Microvessels(WL) P-gp(a) 1 NTTGALTTR BLQ(<1.64) BLQ(<1.64) BLQ(<1.64) 15.3 ±0.21 P-gp(a) 2 LANDAAQVK 1.02 ±0.02 BLQ(<1.02) 1.22 ±0.10 23.8 ±0.32 P-gp(a/b) 3 IATEAIENFR 1.59 ±0.00 BLQ(<0.752) 1.04 ±0.09 17.9 ±0.07 Bcrp 1 SSLLDVLAAR 30.6 ±1.32 1.49 ±0.11 BLQ(<0.751) 0.854 ±0.00 Bcrp 2 VGTQFIR 28.4 ±1.33 1.35 ±0.06 BLQ(<0.567) BLQ(<0.567) Atp1a1/2/3 0 AAVPDAVGK 150 ±3.32 16.4 ±0.01 32.8 ±3.39 213 ±2.90 Atp1a1 0 IVEIPFNSTNK 134 ±3.82 11.1 ±0.40 5.06 ±0.34 32.9 ±0.54 Atp1a2 0 GIVIATGDR BLQ(<0.75) BLQ(<0.75) 3.49 ±0.05 22.1 ±1.13 Atp1a3 0 GVVVATGDR BLQ(<0.961) BLQ(<0.961) 11.3 ±0.52 68.1 ±3.07

Thevaluespresentedwerecalculatedasthemean(±sd.)oftwodigestionreplicates,eachsurveyedwith3or4MRMtransitions;exceptforAAVPDAVGK,thatwasquantified usingonly2transitions.Valuesbelowthelimitofquantification(BLQ)weredeterminedusingtheLOQsobtainedfromtheyeastdigestmatrix(showninparenthesis). VGTQFIRisnotpresentintheratBcrpsequenceandwasnotdetected(ND)inthesesamples.PMP=plasmamembraneproteins,WL=wholelysate.

neitherinthelight-to-heavypeptideratiolevelnorwhen

quantifi-cationwasperformed.Similarly,mousekidneyPMPdigests(n=3)

spikedwiththeheavypeptideswereinjectedthreetimestoverify

thepeptidestabilityinsampleafter45and75h(TableS-10).No

differencewasseeninthelight-toheavyratioorthedetermined

abundanceforanyofthepeptidesquantifiedinmouse.Thus,our

methodallowsthepeptidequantificationwithoutsignificantbias

forlargesamplesets.Inanycase,QCsshouldbeinjectedatdifferent

timesoftheanalysistochecktheaccuracyandprecision.

3.3. Applicationofthemethod-AQUAquantificationofP-gp,

BcrpandNa+_/K+_ATPase

Thedeveloppedandvalidatedmethodwasusedtoquantity

P-gp,BcrpandNa+_/K+_{ATPase␣-subunit(Atp1a)isoformsinmouse}

kidney,mousecorticalvesselsandratcorticalmicrovessels.Fig.5

showchromatogammsobtainedafter5␮Linjectionofsamples.The resultsoftheMRMpeptidequantificationinthesamplesare pre-sentedinTable4(valuespertransitionarepresentedinTableS-11). Thequantitativevalueswerecalculatedasthemean(±s.d.)of6 to8analyticalmeasuresfrom3to4MRMtransitions(exceptfor AAVPDAVGK,quantifiedwith2transitions)detectedin2digestion replicates;thereforetheydonottakeintoaccountbiological vari-ability.Thevariabilitybetweenthetransitions waslowforeach sample;presentinggenerallyCVsbellow20%.Noisiytransitions thatelevetadtheCVwerenottakenintoaccountforthe quantifi-cationofthesample(TableS-11).

InSprague–Dawley(SD)ratcortocialmicrovessels,the abun-dance of the IATEAIENFR and NTTGALTTR peptides were only slighlty different, 17.9±0.07 and 15.3+0.21fmol·␮g−1, respec-tively.Thesevaluesarecomparabletothosepreviouslyobtained by Hoshi et al. [2]. in SD and Wistar rat brain (19.0±2.0 and 24.9±1.1fmol·␮g−1 respectively)usingtheNTTGALTTRpeptide. Nevertheless,weobserved a1.5-foldhigherabundanceof LAN-DAAQVK(23.8±0.32fmol·␮g−1_{),whichcouldbeduetodifferences} inthedigestionreleaseofthepeptide.Themousecorticalvessels presented aP-gp abundanceof 1.22±0.10 and1.04±0.09 with LANDAAQVKandIATEAIENFR,respectively,whileNTTGALTTRwas BLQ.Theabundanceswereconsiderablylowerthaninratcortical microvessels,probablybecauseofthepresenceofdifferencesin vesselsisolationmethod.P-gpinplasmamembraneofddymice renalcortex,renalmedullaandliverwasbelowthelimitsof quan-tificationinthestudyofKamiieetal.usingthepeptidesNTTGALTTR and ATVSASHIIR.WealsoobtainedBLQvaluesinC57BL/6 mice liverPMPwiththethreepeptides,probablybecauseitisexpressed in very low levels [1]. In mousekidney PMPwe also obtained BLQ values using NTTGALTTR, but the more sensitivepeptides LANDAAQVKandIATEAIENFRhadabundancesof1.02±0.02and 1.59±0.00fmol·␮g−1,respectively.

These inter-peptide abundances differences observed could be due to isoformsor differences in digestion release. Indeed, IATEAIENFRispresentinthetwoP-gpisoformsofP-gp(Abcb1a andb)expressedinrodents.Inratkidneyandliverbothisoforms arefound[29]anditcouldbeexpectedthattheisoformspecificity inmouseshouldbesimilar;thusthe0.57fmol·␮g-1higher abun-danceinIATEAIENFRcouldrepresenttheMdr1bisoform.Thisdo notexplainthedifferencesinratcorticalmicrovesselsandmouse corticalvessels,asithasbeenfoundinratcorticalmicrovesselsis almostexclusivelytheAbcb1aisoform[19].Therefore,the1.3-fold higherlevelsofLANDAAQVKthanIATEAIENFR(inbothanimals) and1.5-foldhigherthanNTTGALTTR(inrat)couldbedueto dif-ferentdigestionreleaseofthepeptides,aspreviouslysuggested [15].

BcrpwasBLQinmousecorticalvessels,butinSDratcortical microvesselstheabundanceofSSLLDVLAARwas0.854fmol·␮g−1_. This waslower than measuredby Kamiee et al.[1] (using the

samepeptide),whichcanbeexplainedbyapossible heterogene-ityofthesamplebetweenthetwostudies.IndeedBcrpunlikePgp isexpressedindifferentcellsofthebrainsuchaspericytesand astrocytes [30].Theanalyzed samplesinthetwostudiesmight not havethe samecellcomposition andsooverexpressed Bcrp inKamiieetal.study.TheabundanceofSSLLDVLAARand VGTQ-FIRinmousekidneywere30.6±1.32and28.4±1.33fmol·␮g-1, whichissimilarmagnitudestolevelsobtainedbyKamiieetal.[1] (56.4±1.82and25.9±1.35fmol·␮g-1inrenalcortexandmedulla, respectively).Inmouseliverweobtainedabundancesof1.49±0.11 and1.35±0.06fmol·␮g−1.Inthesetissues,thelevelsofbothBcrp peptideswereverysimilar;althoughtheslightlylowervaluefor VGTQFIRcouldbeduetoslightdegradation,asshowninthe diges-tionstabilitysection.Inageneralway,P-gpwasfoundinhigher levelsthanBcrpintherodentscorticalvesselswhileinmouse kid-neyitisthecontraryasBcrpisalmost30-foldmoreexpressedthan P-gp.

Na+_/K+_{ATPase␣-subunit(ATp1a)wasquantifiedasamarker} oftheplasmamembrane.Thepreviouslyreportedpeptide AAVP-DAVGK[1]commontoisoformsa1,a2anda3wasusedforthe quantificationasamulti-isoformprobe,butwealsoselected pep-tidesspecificforeachoneoftheseisoforms.Thea1-isoformpeptide (IVEIPFNSTNK) wasdetectedinallthetissuesanalysed, butthe a2anda3peptides(GIVIATGDRandGVVVATGDR)werequantified onlyinbraincorticalvessels.Thisisinaccordancewithprevious studieswhichshowedthatrodentliverandkidneyexpressalmost exclusivelythea1isoformwhileinnervoustisues thea3isthe major isoformand a2can alsobefound[26]. Interestingly,we observedthatthea3amountis2-foldhigherthana1and3-fold higherthana2inmousecorticalvesselsandratcortical microves-sels.Inthesesamples,thetotalamountofatpa1,a2anda3peptides correspondto60% oftheamountof AAVPDAVGK;which could beduetodifferentdigestionrelease,butmorestudiesshouldbe performedtoverifythishypothesis.

Theabundancedifferencesbetweenpeptidesofasameprotein thatweobservedpointouttheimportanceoftheselectionof sur-rogatepeptidesforLC–MS/MSproteinquantification,asdifferent probesmaybiastheresults.Therefore,wheneverpossible,several peptideprobesshouldbeusedforthequantificationofeach pro-teintoincreasethecertaintyonthemeasuredvalues.Inaddition, itisadvisabletousethesamepeptideprobeswhenproteinlevels arecomparedbetweensamplesorexperimentalconditions.

4. Conclusion

WedevelopedamethodforthequantificationofP-gp,Bcrpand Na+_/K+_{ATPase␣-subunitisoformsattheBBBbyLC–MS/MS(MRM)} usingtheAQUAstrategywithseveralpeptidesperprotein.Allthe assayshavebeencomprehensivelyvalidatedintermsoflinearity, accuracy,precision,digestionefficiencyandpeptidestability.This methodwassuccessfullyappliedtothedeterminationofproteins inratmousekidney,mousecorticalvesselsandratcorticalmicro vessels. Thedifferentlevelsobtainedforeach peptidehighlight theimportanceanddifficultyofchoosingsurrogatepeptidesfor proteinquantification.

Authorcontributions

DGZ,PS,MV,MP,IB,ICG,JMS,YP,XDandMCMparticipatedin researchdesign.DGZ,MT,MS,WQ,CC,MP,ETandMCMperformed experiments.DGZdevelopedthequantitativeMSmethod.DGZand MCMperformeddataanalysis.ThemanuscriptwaswrittenbyDGZ andMCMwithcontributionsandapprobationfromallauthors.

Acknowledgements

ThisworkwaspartlyfinancedbyServierLaboratories(Orléans, France).TheauthorswouldliketoacknowledgeStéphanie Chas-seigneauxforherhelpinthepreparationofmousecorticalvessels, CatarinaChavesforkindlyprovidingusratcorticalmicrovessels proteinsandFranc¸ois Guillonneauofproteomicsplatform(3P5, ParisDescartesUniversity)forMALDIspectra.

AppendixA. Supplementarydata

Supplementarymaterial relatedto this articlecanbe found, intheonlineversion,atdoi:https://doi.org/10.1016/j.jpba.2018.11. 013.

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