Liquid extraction surface analysis for native mass spectrometry: Protein complexes and ligand binding

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Contents lists available atScienceDirect

International

Journal

of

Mass

Spectrometry

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 / i j m s

Liquid

extraction

surface

analysis

for

native

mass

spectrometry:

Protein

complexes

and

ligand

binding

Victor

A. Mikhailov

a,1

_,

_Rian

_L.

_Grifﬁths

b,1

_,

_Helen

_J.

_Cooper

b,∗

a_Department_of_Chemistry,_University_of_Oxford,_Oxford,_OX1_3TA,_UK

b_School_of_Biosciences,_University_of_Birmingham,_Edgbaston,_Birmingham_B15_2TT,_UK

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received26May2016

Receivedinrevisedform1September2016 Accepted20September2016

Availableonlinexxx Keywords:

Liquidextractionsurfaceanalysis(LESA) Nativemassspectrometry

Non-covalentproteincomplexes Protein-ligandinteractions Drugbinding

a

b

s

t

r

a

c

t

Nativeliquidextractionsurfaceanalysis(LESA)massspectrometryenablesthedirectsamplingof pro-teincomplexesfromasolidsurface.WehavepreviouslydemonstratednativeLESAmassspectrometry ofholomyoglobin(∼17kDa)fromglassslidesandtetramerichaemoglobin(∼64kDa)fromdriedblood spotsandthintissuesections.Here,wefurtherexplorethecapabilitiesofthisemergingtechniqueby investigatingarangeofproteinswhichexistinvariousoligomericstatesinvivo.Tetramericavidin (∼64kDa),octameric(∼190kDa)andhexadecameric(∼380kDa)CS2 hydrolase,and tetradecameric

GroEL(∼800kDa)werealldetectedbynativeLESAmassspectrometry.Moreover,trimericAmtB,a mem-braneprotein,couldalsobeobservedbynativeLESAmassspectrometry.ThesuitabilityofLESAmass spectrometryforprobingprotein-ligandbindingwasalsoinvestigated.Non-covalentcomplexesofthe ligandbiotinwiththeproteinsavidin,haemoglobinandbovineserumalbuminweredetected.Theresults indicatethatnon-speciﬁcbindingisminimalandthatnativeLESAmassspectrometryisapromisingtool fortheinvestigationofbiologicallysigniﬁcantligandbinding.

1. Introduction

Knowledgeofproteintertiaryandquaternarystructureis gen-erallyrequiredforunderstandingproteinfunctionormalfunction. Experimentalmethodsthatprovidethisknowledgearecapableof maintainingnoncovalentbonds,suchashydrogenbondsand␲-␲ interactions,thuspreservingfoldedproteinsintheirnative struc-tureandthenoncovalentbondingwiththosespecieswithwhich theyinteract.Nativeelectrospraymassspectrometry(MS)enables analysisoffoldedproteinsandnon-covalentproteincomplexesin thegasphasebychoosinga non-denaturingelectrospraybuffer andcarefuloptimisationofelectricﬁeldsandpressuresinthemass spectrometer[1–4].IncombinationwithotherMStechniques,such asionmobilitymeasurements[5–7],gas-phasedissociation meth-ods[8,9],hydrogen-deuteriumexchange[10–12]andnon-covalent labelling[12–14]morespeciﬁcinformationontheoverall topol-ogyof non-covalent complexes, connectivity betweendifferent subunitsinthecomplexanddrugbindingsitescanbeobtained. Morerecently,anativeMSapproachhasbeendevelopedfor mem-braneproteincomplexes[15,16].Stabilisationofthesecomplexes

∗ Correspondingauthor.

E-mailaddress:[email protected](H.J.Cooper). 1 _These_authors_contributed_equally_to_this_work.

insolutionandthegas-phaserequiresthattheyarepuriﬁedand electrosprayedindetergentmicelles.ThenativeMSapproachalso allows investigationof interactions of membrane protein com-plexeswithlipidsanddrugs[17,18].

Nativemassspectrometryischaracterisedbyionsinrelatively lowchargestates–fewerprotonationsitesareavailableinfolded proteins–andthushighermass-to-chargeratiosarerecorded(for gas-phaseproteincomplexestypicalm/z_≥3000)[1,2].Increasing theupper limit ofthemass range toprobe everlargerprotein assemblieshasbeenoneofthemajormotivesindevelopmentofthe nativeMSapproach.Q-TOFmassanalysershavebeentheanalysers ofchoiceinnativeESI,duetotheirhighm/zrange[2,19];however, morerecentlynativeMShasbeensuccessfullyimplementedon FTICRandOrbitrapinstruments[20,21],whichoffermass resolu-tionsuperiortoQ-TOF.Todate,massspectraofintactcomplexes aslargeas18MDa (anentire capsidassembly ofbacteriophage HK97)havebeenreported[22],althoughthemassresolutionof suchlargespeciesislimitedbyincompletedesolvationoftheir gas-phaseionsratherthanbytheinherentinstrumentresolutionofthe massanalysersemployed[23].

MostnativeMSexperimentsarecarriedoutbydirect electro-sprayinfusionofthepuriﬁedsampleintothemassspectrometer. AnalyticalapplicationsofthenativeMSapproachcanbeexpanded considerablywithsamplingofnon-covalentprotein–proteinand protein-ligandcomplexesdirectlyfromsurfaces:theavenuesfor http://dx.doi.org/10.1016/j.ijms.2016.09.011

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future explorations include probing diverse biological samples, suchasdriedbloodspotsandthintissuesections,protein/ligand assays and biomimicking membranes. Surface samplingcan be coupledto electrospray ionisation (essential for native MS) by desorptionelectrosprayionisation(DESI)orliquidmicrojunction basedtechnologiessuchasliquidextractionsurfaceanalysis(LESA) [24,25].InDESI,ajetofelectrosprayedsolventisdirectedata sur-facecontaininganalytemoleculesinordertodesorbanddeliver themintothemassspectrometer[26–28].Thistechniqueproduces multiplychargedionssimilartodirectESIinfusion[29,30].DESIhas beensuccessfullyappliedtothesurfaceanalysisofsmallmolecules, peptidesandproteinsupto∼25kDa [27,31,32],however direct analysisofintactproteinsfrombiologicallyrelevantsampleshas notbeenreportedtodate.AvariantofDESIinwhichliquid sam-plesareprobed(liquid-DESI)hasdemonstratedthatintactproteins andproteincomplexesupto∼150kDacanbedetected[29].

Inanotherapproach,DESIcanbeusedtoprobenon-covalent interactions between immobilisedproteins and ligands. In this methodproteinsareimmobilisedonasurfaceandtreatedwitha mixtureofligands.Thenon-bindingligandsarethenwashedaway, andtheassayissubjectedtoDESIanalysiswithdenaturingsolvents leadingtothedetachmentof theboundligandmoleculesfrom theproteinandtheirdeliverytothemassspectrometer[33].This approachcanbeusedforhigh-throughputscreeningforbinding ligands,buttheindirectassessmentofnon-covalentinteractions betweentheproteinandtheligandsviadetectionoftheligand moleculesbyDESI is not entirelyconclusive,becausethe pres-enceoftheimmobilisingagentcanproducefalsepositiveresults [34].Furthermore,these studiescannotprovide informationon thestoichiometryofbinding,and arenotapplicabletosamples withheterogeneousproteintargets,suchasbloodspotsandtissue sections.

Alternativesurface sampling techniques that are potentially capableofextractingadiverserangeofnon-covalentcomplexesfor MSanalysisareliquidmicrojunctionbasedmethodssuchasliquid extractionsurfaceanalysis(LESA)[24,25]orcontinuousflow liq-uidmicrojunctionsampling[35].LESAenablessamplingofdiscrete locationsonasurfacebyplacingasmalldropletofthesolventona spotwhichisthenre-aspiredwithaconductivepipettetippriorto ESIMS.LESAMSanalysisofintactproteinshasbeenachievedfrom avarietyofsubstrates:driedbloodspotsonfilterpaper[36–39], thintissuesectionsthaw-mountedontoglassslides[40–43]and E.colicoloniesgrownonagargel[44].Recently,proteinspeciesup to∼15kDawerereportedby‘nanoDESI’imagingoftissuesections [45].Despiteitsname,nano-DESIisactuallybasedoncontinuous flowliquidmicrojunctionsurfacesampling[46].

We recently reported the use of LESA with non-denaturing ammoniumacetatebasedsolventfortheMSanalysisof nonco-valentproteincomplexesfromglassandpolyvinylideneﬂuoride (PVDF) substrates [47]. Using this ‘native’ LESA MS method, intact∼17.5kDaholomyoglobin,anon-covalentmyoglobin-heme complex, and a range of non-covalently bound species from a haemoglobinsample:␣H_and_␤H_-globin_subunits_associated_with

heme,∼16kDaeach;the(␣␤)2H_heterodimers₍_∼32_kDa)_and_the

intact(␣␤)2 4H tetrameric complexes(∼64kDa)weredetected.

Furthermore,theintacthaemoglobintetramercouldbedetected fromdriedbloodspotsonﬁlterpaper.Grifﬁthsetal.subsequently demonstrated direct detection of the tetrameric haemoglobin complexfromvasculaturein thintissuesectionsofmouseliver thaw-mountedontoglassslides[48].

Intheworkpresentedhere,wesoughttofurthercharacterise thecapabilitiesofnativeLESAMS.Wedemonstrateasigniﬁcant extensionofthemassrangeofourmethod,and showthatit is possibletodetectintactproteinassembliesaslargeas_∼800kDa. Wealsodemonstratethatmembraneproteincomplexes,thatare preparedinmicelle-containingsolutions,areamenabletonative

LESAMSandcanbeprobeddirectlyfromdriedspotsonasurface. ThemassspectraproducedbynativeLESAMSaresimilartothose obtainedfrom‘standard’nativemassspectrometry.Furthermore, protein-ligandinteractionshavebeenprobedbyLESAMS,andthe detectionofbiotinnoncovalentlyboundtoavidin,haemoglobin andBSAispresented.

2. Materialsandmethods

2.1. Materials

Protein standards (avidin from chicken egg white, bovine serumalbumin(BSA),humanhaemoglobin,Chaperonin60from Escherichiacoli(GroEL)),biotinandammoniumacetatewere pur-chasedfromSigma-Aldrich(Gillingham,UK). GroELwasfurther puriﬁed as described elsewhere [1]. CS2 hydrolase from

Acidi-anusA1-3wasagiftfromJasminMecinovic,RadboudUniversity Nijmegen, Netherlands [49]. Ammonium channel (AmtB) was expressedand puriﬁed in-houseat theUniversity of Oxford as describedelsewhere[50].Octyltetraglycol(C8E4)waspurchased fromAvanti(Alabaster,AL,USA).

2.2. Samplepreparation

Proteins were prepared in 200mM ammonium acetate (pH=7.5) resulting in a ﬁnal protein concentration 1–10_␮M. GroELandBSAproteinswerefurtherdesaltedusingMicro-BioSpin columns(BioRad,Hercules,CA,USA).AmtBwaspreparedin ammo-niumacetatesolutioncontainingC8E4attwicethecriticalmicelle concentration.1␮lofproteinsolutionwasspottedontoacleaned glassslideforLESA.Thesamplespotswereair-driedatroom tem-peratureandprobedusingLESAwithadelayof20–90minbetween thedepositionofthesampleanditsextractionforMSanalysis. 2.3. Surfacesampling

LESAwascarriedoutusingtheTriversaNanomatechip-based ESI source (Advion Biosciences, Ithaca, USA) coupled with the SynaptG2Smassspectrometer(Waters,Manchester,UK).The sam-plewasmounted ontotheLESAuniversaladaptorplateandan imagewasacquiredusinganEpsonPerfectionV300photoscanner. TheexactlocationtobesampledwasselectedusingtheLESAPoints software(Advion).Theextraction/ionisationsolventwas200mM ammoniumacetateinwater(HPLCgrade,JTBaker,Deventer,The Netherlands).Forbiotinbindingstudies,theammoniumacetate solutionwasusedtospottheproteinsamplesontheslide,the extractionsolutioncontainedeither1.0or0.1mMbiotinin200mM ammoniumacetate.Intheextractionprocess,thesolventwas aspi-ratedfromthesolventwell,beforetheroboticarmrelocatedtoa positionabovethesampleslide.Someofthesolventwasthen dis-pensedandaliquidmicrojunctionwasmaintainedbetweenthe pipettetipand thesamplesurfacebeforeaspiratingthesolvent anddeliveringittotheelectrospraychip.Thedistancebetweenthe nanospraychipandtheentrancetothesampleconewas3–6mm. ThedetailsoftheLESAextractionprocedureandionsource (Nano-mate)parametersforeachproteinaregiveninTable1.

2.4. Massspectrometry

Aspeedivalve(BOCEdwards,Crawley,Sussex,UK)wasinstalled betweentherotaryand turbopumpsthat pumptheion source regioninSynaptG2S(Waters,Wilmslow,UK).Thepressureinthe ionsourcecanbecontrolledbycarefulthrottlingofthepumpingby thespeedivalvewiththeaimofimprovingthetransmissionofions withhighm/zthatarecharacteristicforintactproteincomplexes.

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Table1

ParametersofLESAextractionprocedureandionsourceconditions.

Protein Volumeaspirated

fromsolventwell (␮l)

Heightabovethe surface(mm)

Volume dispensedonto thesurface(␮l)

Delaytime(secs) Volume reaspirated(␮l) Gaspressure (psi) ConeVoltage (kV) AmtB 2.0 0.8 1.5 60 2.0 0.3 1.9 CS2hydrolase 2.0 0.8 1.5 60 2.0 0.3 1.9 GroEL 4.0 0.6 2.0 30 2.5 0.15 1.8 Haemoglobin 4.0 0.6 2.0 30 2.5 0.15 1.8 Avidin 4.0 0.6 2.0 30 2.5 0.15 1.8 BSA 4.0 0.6 2.0 30 2.5 0.15 1.8

Thebackingpressurewasalteredfrom3to9mbarforthese

exper-iments.MassspectrawereacquiredinthefullscanTOFmodeon

theSynaptG2S.Thescantimewassetat2–5s,them/zrangewas

altereddependingonthespeciesinvestigated(usually≥10,000).

Thecapillarytemperaturewassetto30◦Candtheconevoltagewas

setat45V.Insomeexperiments(seetext)collisioninduced

disso-ciation(CID)wascarriedoutontheionsinthetrapregionofthe

massspectrometer.Collisionvoltagewasvariedfrom50to200V.

AlldatawereanalysedbyMassLynxsoftware(version4.1,Waters).

Therawmassspectrahavebeenaveragedfor20–100scansand

presentedherewithminimalsmoothingandbackground

subtrac-tion.ThenativeLESAmassspectrahavebeencomparedagainst

themassspectraofthesameproteinsusingdirectinfusion

elec-trospraywithNanomateonthesamemassspectrometerandthe

massspectrafromthepreviouswork.Forselectedexamples(e.g.

avidin-biotinbinding),LESAMSspectrawerecomparedwith

high-resolutionnativeMSspectraobtainedonanOrbitrapQ-Exactive

instrument(Thermo,Bremen,Germany)intheUniversityofOxford

modiﬁedforhighm/zrange,similartothemodiﬁcationspreviously

reportedbyRoseetal.[21].

3. Resultsanddiscussion

3.1. Protein-proteincomplexes

Arangeofproteinswhichexistinoligomericstatesinvivoand innon-denaturingsolutionswereinvestigated.Theirnon-covalent complexesvaryinmassfrom∼60kDaupto∼800kDaandexistin differentoligomericstates,fromtrimers(AmtB)tohexadecamers (CS2 hydrolase).Fig.1Ashowstheresultsobtainedforavidin, a

solubletetrameric glycoprotein,which hasbeenlong-usedas a standardfor nativeMS[51].Themassoftheavidintetrameris similartothatofthehaemoglobintetramerdescribedinLESAMS studiesin ourpreviousworks [48,52].Sampling thedried pro-teinonglasswith200mMammoniumacetatesolutionresulted in the detection of thetetrameric species (∼64kDa) in charge statesrangingfrom12+to16+ atm/z∼5300,4900,4600,4300 and4000respectively,andareinaccordancewiththechargestate distributionusuallyobservedinthemassspectraofavidinfrom non-denaturingelectrospray[53].Thetetramerpeaksshowninthe spectrumarerelativelybroad(∼200Th).Usuallypeakbroadening innativeMSiscausedbyincompletedesolvationofthegas-phase molecularionsandthepresenceofsalts[47],butcanalsobeasign ofproteindegradation.Ahigh-resolutionmassspectrumofavidin, producedbydirectnano-electrosprayonanOrbitrapQ-Exactive massspectrometerthatwasmodiﬁedforhighm/zrange,shows thepresenceofpeakscorrespondingtothesemodiﬁcationswithin theenvelopofeachchargestateofthetetramer(Fig.S1A, Support-inginformation).Massspectraofthechargestates(14+to18+)of theavidintetramerobtainedontheNanomate/SynaptG2S appa-ratusbydirectinfusiondemonstratepeakbroadeningsimilarto LESAMS(Fig.S1BversusFig.1A).Furthermore,collisioninduced dissociation(CID)oftheavidintetramerrevealsmultiplepeaksfor eachchargestateofthefragmentmonomer(Fig.S1B),indicating

thepresenceofmultiplemodiﬁcationsontheproteinbackbone. TheseresultssuggesttheavidintetramerionsobservedinLESA MShavethesamestructureasthoseobservedin‘standard’native MS.Furtherevidencethatavidinretaineditsbiologicallyrelevant stateintheLESAMSapproachcamefromourresultsforitsbinding tobiotin(seebelow).

Carbon disulphide hydrolase (CS2 hydrolase) is an enzyme

that converts carbon disulphide to carbon dioxide and hydro-gensulphide.Itexistsasa190kDaoctamericringanda380kDa hexadecamericcatenane(locked-ringstructure).Thecatenane for-mationwasreportedasbiologicallyrelevantin2013byEldijketal. [54].Surfacesamplingofdriedsolutionsofthisproteinledtothe detectionofboth oftheseoligomericforms.Theoctamericring structurewasdetectedinchargestatesrangingfrom26+to31+ betweenm/z6000to8000asshowninFig.1B.Inthesamemass spectrum,thehexadecamericcatenaneisrecordedinchargestates between35+to42+(m/zrange9000–12000).Similarmassspectra oftheCS2hydrolasewerereportedinearlierdirectinfusionnative

MS[49],andwereobtainedinthiswork:directinfusion nanoelec-trosprayofthesameproteinsolutionusedforLESAMSresulted inthemassspectrumshowninFig.S1C.Thefactthatboth bio-logicallyrelevantoligomericformsofCS2hydrolasesurvivedthe

dryingstageoftheprocedureandcouldthenbedetectedbyLESA MSemphasisesthebiologicalrelevanceofthismethod.

The largestsoluble complexanalysed herebyLESA MS was GroEL,achaperoneproteinwhichformsa∼800kDatetradecameric complexconsistingoftworingheptamers.GroELtetradecameris asolubleproteincomplexwhichhasoftenbeenusedtotestthe uppermassrangeinnativeMSapplications[21,55–57].GroELwas detectedbyLESAMSfromdriedproteinsamplesinchargestates ranging from61+ to68+ at m/z∼13100,12900, 12750, 12500, 12350,12200,12000and 11850respectively(Fig.1C).Previous nativeelectrosprayionisationstudiesinammoniumacetatebased solventsystemshave reportedthedetectionof theGroEL com-plexinthesimilarm/zrangewithslightvariationsinthecharge statedistributionthatwasapparentlydependentonthetypeof mass spectrometerused [21,56,57]. We also detectedunfolded monomeric subunits of GroELat m/z<4000(∼58kDa, datanot shown),whichmightbeanindicationofeitherpartialdegradation ofthecomplexinthedryspotsbeforeLESAextraction,orcollision induceddissociationintheionsource.Nevertheless,mostofthe GroELtetradecamerwasretainedduringtheLESAprocedureand producedmassspectrasimilartonativeMSdirectlyfromsolution, indicativeofthesimilarityofthegas-phaseGroELionsformedin thesetwomethods.

ThenativeLESAMSapproachwasalsoappliedtoa different classof proteincomplexes:membrane proteins.The encapsula-tionofhydrophobicmembranecomplexesindetergentmicelles isrequiredtoretaintheirstabilityinsolutionandthegasphase. NativeESImassspectraofmembraneproteincomplexesinmicelles result inunstructured features due totheheterogeneity of the micelles.Collisionactivationisusedtoliberatethemembrane com-plexfromthegas-phasemicellesandresolveitschargestates[16]. WeusedasimilarapproachinourLESAMSexperiment.AmtBis

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Fig.1.NativeLESAmassspectraofproteincomplexesfromglasssubstrate.A)Massspectrumofavidintetramer(64kDa)aftercollisionactivationofavidinionsinthetrap region(100Vcollisionvoltage).

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Fig.2. NativeLESAmassspectraofBSAfromglasssubstrate.A)BSAspotssampledwith200mMammoniumacetate.B)BSAspotssampledwith200mMammonium acetate/1mMbiotin.

amembranetransporterproteinwhichformsatrimericcomplex withamassofapproximately140kDa.AmtBinC8E4micellesin ammoniumacetatesolutionwasspottedontheglassslidesand, afterthespotsweredried,wasextractedandanalysedbyLESAMS usingpureammoniumacetatesolution.Toresolvethechargestates ofthis trimercomplex weusedanelevatedcollisionvoltageof 200VinthetrappartoftheSynaptG2S.Wedetectedchargestates oftheintacttrimerrangingfrom17+to20+atm/z∼8400,7900, 7400and7000respectively.Thesepeakswererelativelynarrow, ≤50Thwide(Fig.1D),incomparisonwiththewidthofthepeaks ofthesolublecomplexes(Fig.1A–C).Thiseffectisprobablydueto thefactthatthemembraneproteinisnotlikelytoretainmany sol-ventmoleculesbecauseofitslimitedhydrophilicsurfaceandthe largeactivationenergythathadtobeappliedtoitsgas-phaseions inordertoremovethedetergentmoleculesandrevealthecharge states.AsimilarmassspectrumofAmtBtrimerwasproducedby directinfusionnanoelectrospray(Fig.S1D).

3.2. Protein-ligandinteractions

Biotin(244Da)bindstoavidinspecificallyandwithveryhigh affinity(onebiotinmoleculepereachavidinmonomer).This prop-ertyisexploitedinbiotechnologytoisolatebiotinylatedmolecules inaffinityassaysorchromatography,whereimmobilisedavidinor homologousproteinsareusedtoanchorthemoleculesofinterest [58].Asbiotinbindingisfast,asolutionofbiotininammonium acetatewasusedtosampledriedspotsofavidinbyLESAinorder totestwhetherthebindingtoavidincantakeplaceduringthis procedure. Samplingwith 200mM ammonium acetatesolution containing1mM biotinledtoamass shiftforthemajorpeaks ofthechargeofavidintetramer,asshowninFig.1Ainset.This massshift,averagedfor14+to16+chargestate,relatestoan addi-tionof∼960Da,indicatingthatasmanyasfourbiotinmolecules (244Da×4=976Da)areboundtotheavidintetramer.Atpresent wearelimitedbytheresolvingpoweroftheSynaptmass spectrom-eterandtherelativelysmallm/zrangeoftheOrbitrapcoupledto LESAinBirmingham(m/z<4000)todemonstratespecificbinding ofbiotintoavidintetramerwithhigherresolutionbythismethod. WehavealsotestedbiotinbindingtoavidinontheOrbitrap Q-Exactive(upperm/zlimit30,000Th,UniversityofOxford)bythe directinfusionofavidin/biotinsolutionusingananospray capil-lary,Fig.S2AandB.Asthefinestructureofthechargestatesof avidincanberesolvedontheOrbitrap,themassshiftofthepeaks correspondingtobiotinbindingwascalculatedtobeexactly976Da (fourbiotinmoleculesbound).Moreover,CIDoftheavidin/biotin non-covalentconjugateat100Vcollisionvoltageresultedinthe productionofapo-avidintetramer(−976Damassshift,Fig.S2C). Therefore,weconcludethatimplementingLESAMSona

higher-resolution/highermassrangeinstrumentinfutureshouldbeable toresolvetheﬁnestructureofmultiplymodiﬁedproteinsand pro-teincomplexesandtheexactstoichiometryofthedrugbindingto them.

Determinationoftheoverallpharmacokineticprofileofadrug requiresmeasurementsofitsaffinitytowardplasmaproteins[59]. NativeLESAMSwasappliedtotheinvestigationofbindingofbiotin totwobloodproteins,BSAandhumanhaemoglobin,inafashion similartotheavidinexperiments.Thedegreeofmodificationof wildtypeBSAissmallerthanthatofmultiplyglycosylatedavidin. It wasthereforepossibletoresolve peakscorrespondingtothe bindingofindividualbiotinmolecules(atleastuptofour)toBSA (chargestates13+to17+)ontheSynaptG2ScoupledtoLESA,Fig.2, althoughtheresolutionwasnothighenoughtoestimatethe max-imumnumberofboundmolecules.Biotinbindingtohaemoglobin dimers,(␣␤)2H_was_also_detected,_with_better_mass_resolution_and

relativelyhighsignalintensity,inchargestatesrangingfrom12+ to8+betweenm/z2500to4250,Fig.3.Atleastsevenmolecules ofbiotinboundtothehaemoglobindimerweredetectedatzero collisionenergyinthetrapregionofSynaptG2S.Thetetrameric haemoglobin((␣␤2)4H)boundtobiotinwasalsodetectedwitha

lowermassresolutioninthechargestatesfrom14+to16+,and withapositivemassshiftinrespecttothepeaksofthecharge statesofunboundhaemoglobintetramers(datanotshown),but theexactnumberoftheboundbiotinmoleculeswasimpossibleto estimatewiththislevelofmassresolution.Peakscorrespondingto ␣-and␤-globinmonomerandmonomer-hemespeciesincharge statesrangingfrom8+to16+werealsoobserved,butitisnot appar-entinthemassspectrawhetheranybiotinbindingtookplacefor themonomers(Fig.3B):itis,however,possibletoconcludethat apo-proteinmonomericionsaremuchmoreabundantthanthe ionswhichmightbecorrespondingtoprotein/biotincomplexes, sothebindingofbiotintothemonomerichaemoglobincannotbe asstrongastothedimers.Theseresultsareinaccordwitha pre-viouslyreportedstudyonbiotinbindingtohumanbloodplasma proteins[60].Althoughtheauthorsdidnotinvestigatethebiotin bindingtospeciﬁcproteins,theyfoundoutthat12%ofbiotinin plasmaiscovalentlyboundand7%isboundreversibly.

Non-specificbindingofligandstoproteinsornon-specific for-mationofnon-covalentproteincomplexesissometimesobserved innativemassspectrometry[1,2].Inordertoexcludenon-specific bindingfromthemassspectra,theyareoftencollectedeitherina serieswithdiminishingligandorproteinconcentrationsinthe elec-trospraysolutioninordertodecreasethechancesofnon-specific associationin theelectrospraymicrodroplets,orwithextraion activation(e.g.,carefullyelevatedsamplingconeorcollision volt-age)todissociatenon-specificcomplexesinthegas-phase.LESA massspectraofhaemoglobinandhaemoglobin/biotinwitha

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colli-Fig.3.NativeLESAmassspectraofhaemoglobinfromglasssubstrate.A)andC)Haemoglobinspotssampledwith200mMammoniumacetate.B)andD)Haemoglobinspots sampledwith200mMammoniumacetate/1mMbiotin.C)andD)100Vtrapcollisionvoltagewasused.

sionvoltageof100VinthetrapregionofSynaptG2Sareshown inFig.3C andD.Thesemass spectrademonstrate thatsomeof thebiotinmoleculesremainboundtohaemoglobindimerseven undertheharshactivationconditions.Thisresultindicatesthat atleastsomeofthebiotinbindingtohaemoglobinheterodimers observedbyLESAMSisspecificandthereforebiologically signif-icant.Assumingthatbiotindoesnotbindspecificallytounfolded haemoglobinmonomers,theabsenceofstrongMSsignals corre-spondingtobiotinbindingtothemonomershaemoglobin(Fig.3B andD)alsoindicatesthatnon-specificbindinginourLESA experi-mentswasminimal.

4. Conclusions

Liquidextractionsurfaceanalysis(LESA)ofproteinspecieson glasssubstrateshasbeenshowntobesuccessfulforprotein com-plexesupto∼800kDa(GroEL).Thismassissigniﬁcantlygreater thanpreviouslyreported(_∼64kDa,haemoglobin).Remarkably,the investigatedcomplexescouldsurvivearelativelylongtime,from 20mintooveranhour,ontheglasssubstratebeforetheirextraction

byLESA.TheresultsalsoshowthatnativeLESAMShaspotential forapplicationsinmembraneproteinstudiesthatinvolvetheuse ofstabilisingdetergentmicelles,asdemonstratedbythesuccessful LESAMSdetectionoftheAmtBmembranecomplex.Furthermore, thepotentialofnativeLESAMStoprobeprotein-ligand interac-tionshasbeenshownfortheﬁrsttimewiththebindingofbiotinto avidin,BSAandhaemoglobin.Theresultsobtainedforbiotin bind-ingtoavidinconﬁrmedthat avidinremainedinthebiologically activestateduringLESA.Asourpreviousresultsforhaemoglobin demonstrated[47],improvedmassresolutioncanbeachievedby interfacingthismethodwithmassspectrometersofhigher resolv-ingpower,e.g.Orbitrap.

Insummary,nativeLESAmassspectrometryis(1)suitablefor theanalysisofhighmassproteincomplexes;(2)interrogating com-plexesofboth solubleandmembraneproteins;and(3)probing protein-ligandinteractions.Theresultsarehighlyencouragingfor futureattemptstoextendtheuseofthenativeLESAMSmethod totheanalysisof protein/ligand bindingassays, theanalysisof membraneproteincomplexesfrombiomimickingmembranes,and

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insituanalysisofheterogeneousbiologicalsurfaces,suchasdry bloodspotsandtissuesections.

Acknowledgments

RLG and HJC are funded by EPSRC (EP/L023490/1). Waters Corporation is acknowledged for a Fellowship to VAM. The Waters Synapt G2S mass spectrometer was funded by EPSRC (EP/K039245/1).Supplementarydatasupportingthisresearchis openlyavailablefromtheUniversityofBirminghamdataarchive athttp://ﬁndit.bham.ac.uk/.JosephGaultandDenisShutin(Carol Robinson’sgroup)fromtheUniversityofOxfordareacknowledged forprovidingasampleofAmtBandtheirhelpwithcollectingMS dataontheOrbitrapQ-Exactive.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.ijms.2016.09.011.

References

[1]H.Hernandez,C.V.Robinson,Determiningthestoichiometryandinteractions ofmacromolecularassembliesfrommassspectrometry,Nat.Protoc.2(3) (2007)715–726.

[2]A.J.Heck,R.H.vandenHeuvel,Investigationofintactproteincomplexesby massspectrometry,MassSpectrom.Rev.23(5)(2004)368–389. [3]M.Sharon,StructuralMSpullsitsweight,Science340(6136)(2013)

1059–1060.

[4]J.Marcoux,C.V.Robinson,Twentyyearsofgasphasestructuralbiology, Structure(Lond.,Engl.:1993)21(9)(2013)1541–1550.

[5]Z.Hall,A.Politis,C.V.Robinson,Structuralmodelingofheteromericprotein complexesfromdisassemblypathwaysandionmobility-massspectrometry, Structure20(9)(2012)1596–1609.

[6]A.Politis,etal.,Integratingionmobilitymassspectrometrywithmolecular modellingtodeterminethearchitectureofmultiproteincomplexes,PLoSOne 5(8)(2010).

[7]E.vanDuijn,etal.,Chaperonincomplexesmonitoredbyionmobilitymass spectrometry,J.Am.Chem.Soc.131(4)(2009)1452–1459.

[8]J.L.P.Benesch,Collisionalactivationofproteincomplexes:pickingupthe pieces,J.Am.Soc.MassSpectrom.20(3)(2009)341–348.

[9]M.Zhou,C.Huang,V.H.Wysocki,Surface-induceddissociationofion mobility-separatednoncovalentcomplexesinaquadrupole/time-of-ﬂight massspectrometer,Anal.Chem.84(14)(2012)6016–6023.

[10]T.E.Wales,J.R.Engen,Hydrogenexchangemassspectrometryfortheanalysis ofproteindynamics,MassSpectrom.Rev.25(1)(2006)158–170.

[11]J.R.Engen,Analysisofproteinconformationanddynamicsby

hydrogen/deuteriumexchangeMS,Anal.Chem.81(19)(2009)7870–7875. [12]L.Konermann,X.Tong,Y.Pan,Proteinstructureanddynamicsstudiedby

massspectrometry:H/Dexchange,hydroxylradicallabeling,andrelated approaches,J.MassSpectrom.43(8)(2008)1021–1036.

[13]K.Takamoto,M.R.Chance,Radiolyticproteinfootprintingwithmass spectrometrytoprobethestructureofmacromolecularcomplexes,Annu. Rev.Biophys.Biomol.Struct.35(2006)251–276.

[14]Y.Pan,etal.,Structuralcharacterizationofanintegralmembraneproteinin itsnaturallipidenvironmentbyoxidativemethioninelabelingandmass spectrometry,Anal.Chem.81(1)(2009)28–35.

[15]N.P.Barrera,etal.,Micellesprotectmembranecomplexesfromsolutionto vacuum,Science321(5886)(2008)243–246.

[16]A.Laganowsky,etal.,Massspectrometryofintactmembraneprotein complexes,Nat.Protoc.8(4)(2013)639–651.

[17]A.Laganowsky,etal.,Membraneproteinsbindlipidsselectivelytomodulate theirstructureandfunction,Nature510(7503)(2014),p.172-+.

[18]J.Marcoux,etal.,Massspectrometryrevealssynergisticeffectsof nucleotides:lipids,anddrugsbindingtoamultidrugresistanceefﬂuxpump, Proc.Natl.Acad.Sci.U.S.A.110(24)(2013)9704–9709.

[19]F.Sobott,etal.,Atandemmassspectrometerforimprovedtransmissionand analysisoflargemacromolecularassemblies,Anal.Chem.74(6)(2002) 1402–1407.

[20]H.Li,etal.,Nativetop-downelectrosprayionization-massspectrometryof 158kDaproteincomplexbyhigh-resolutionfouriertransformioncyclotron resonancemassspectrometry,Anal.Chem.86(1)(2013)317–320. [21]R.J.Rose,etal.,High-sensitivityOrbitrapmassanalysisofintact

macromolecularassemblies,Nat.Methods9(11)(2012),p.1084-+. [22]J.Snijder,etal.,Studying18MDavirusassemblieswithnativemass

spectrometry,Angew.Chem.Int.Ed.52(14)(2013)4020–4023.

[23]P.Lossl,J.Snijder,A.J.R.Heck,Boundariesofmassresolutioninnativemass spectrometry,J.Am.Soc.MassSpectrom.25(6)(2014)906–917.

[24]D.Eikel,etal.,Liquidextractionsurfaceanalysismassspectrometry (LESA-MS)asanovelproﬁlingtoolfordrugdistributionandmetabolism analysis:theterfenadineexample,RapidCommun.MassSpectrom.25(23) (2011)3587–3596.

[25]W.Rao,etal.,AmbientDESIandLESA-MSanalysisofproteinsadsorbedtoa biomaterialsurfaceusingin-situsurfacetrypticdigestion,J.Am.Soc.Mass Spectrom.24(12)(2013)1927–1936.

[26]Z.Takáts,etal.,Massspectrometrysamplingunderambientconditionswith desorptionelectrosprayionization,Science306(5695)(2004)

471–473.

[27]Y.-S.Shin,etal.,Desorptionelectrosprayionization-massspectrometryof proteins,Anal.Chem.79(9)(2007)3514–3518.

[28]A.A.Stokes,etal.,Top-downproteinsequencingbyCIDandECDusing desorptionelectrosprayionisation(DESI)andhigh-ﬁeldFTICRmass spectrometry,Int.J.Massspectrom.289(1)(2010)54–57.

[29]C.N.Ferguson,etal.,Directionizationoflargeproteinsandproteincomplexes bydesorptionelectrosprayionization-massspectrometry,Anal.Chem.83 (17)(2011)6468–6473.

[30]P.Liu,etal.,Measuringprotein–Ligandinteractionsusingliquidsample desorptionelectrosprayionizationmassspectrometry,Anal.Chem.85(24) (2013)11966–11972.

[31]K.A.Douglass,A.R.Venter,Proteinanalysisbydesorptionelectrospray ionizationmassspectrometryandrelatedmethods,J.MassSpectrom.48(5) (2013)553–560.

[32]J.M.Wiseman,etal.,Desorptionelectrosprayionizationmassspectrometry: imagingdrugsandmetabolitesintissues,Proc.Natl.Acad.Sci.U.S.A.105 (47)(2008)18120–18125.

[33]C.Yao,etal.,Screeningofthebindingofsmallmoleculestoproteinsby desorptionelectrosprayionizationmassspectrometrycombinedwithprotein microarray,J.Am.Soc.MassSpectrom.26(11)(2015)1950–1958.

[34]D.A.Annis,etal.,Afﬁnityselection-massspectrometryscreeningtechniques forsmallmoleculedrugdiscovery,Curr.Opin.Chem.Biol.11(5)(2007) 518–526.

[35]G.J.VanBerkel,V.Kertesz,R.C.King,High-throughputmodeliquid

microjunctionsurfacesamplingprobe,Anal.Chem.81(16)(2009)7096–7101. [36]R.L.Edwards,etal.,Hemoglobinvariantanalysisviadirectsurfacesamplingof

driedbloodspotscoupledwithhigh-resolutionmassspectrometry,Anal. Chem.83(6)(2011)2265–2270.

[37]R.Edwards,etal.,Top-downproteomicsanddirectsurfacesamplingof neonataldriedbloodspots:diagnosisofunknownhemoglobinvariants,J.Am. Soc.MassSpectrom.23(11)(2012)1921–1930.

[38]R.L.Edwards,etal.,Compoundheterozygotesandbeta-thalassemia: top-downmassspectrometryfordetectionofhemoglobinopathies, Proteomics14(10)(2014)1232–1238.

[39]R.L.Grifﬁths,etal.,Liquidextractionsurfaceanalysisﬁeldasymmetric waveformionmobilityspectrometrymassspectrometryfortheanalysisof driedbloodspots,Analyst140(20)(2015)6879–6885.

[40]J.Sarsby,etal.,Top-downandbottom-upidentiﬁcationofproteinsbyliquid extractionsurfaceanalysismassspectrometryofhealthyanddiseasedhuman livertissue,J.Am.Soc.MassSpectrom.25(11)(2014)1953–1961.

[41]J.Sarsby,etal.,Liquidextractionsurfaceanalysismassspectrometrycoupled withﬁeldasymmetricwaveformionmobilityspectrometryforanalysisof intactproteinsfrombiologicalsubstrates,Anal.Chem.87(13)(2015) 6794–6800.

[42]RianL.Grifﬁths,etal.,LESAFAIMSmassspectrometryforthespatialproﬁling ofproteinsfromtissue,Anal.Chem.UnderRevision(2016).

[43]R.L.Grifﬁths,etal.,Formallithiumﬁxationimprovesdirectanalysisoflipids intissuebymassspectrometry,Anal.Chem.85(15)(2013)7146–7153. [44]E.C.Randall,J.Bunch,H.J.Cooper,Directanalysisofintactproteinsfrom

Escherichiacolicoloniesbyliquidextractionsurfaceanalysismass spectrometry,Anal.Chem.86(21)(2014)10504–10510.

[45]C.-C.Hsu,P.-T.Chou,R.N.Zare,Imagingofproteinsintissuesamplesusing nanospraydesorptionelectrosprayionizationmassspectrometry,Anal. Chem.87(22)(2015)11171–11175.

[46]P.J.Roach,J.Laskin,A.Laskin,Nanospraydesorptionelectrosprayionization: anambientmethodforliquid-extractionsurfacesamplinginmass spectrometry,Analyst135(9)(2010)2233–2236.

[47]N.Martin,etal.,Nativeliquidextractionsurfaceanalysismassspectrometry: analysisofnoncovalentproteincomplexesdirectlyfromdriedsubstrates,J. Am.Soc.MassSpectrom.2015(2016)1–8.

[48]R.L.Grifﬁths,H.J.Cooper,Directtissueproﬁlingofproteincomplexes:toward nativemassspectrometryimaging,Anal.Chem.88(1)(2016)606–609. [49]M.B.vanEldijk,etal.,EvidencethatthecatenaneformofCS2hydrolaseisnot

anartefact,Chem.Commun.49(71)(2013)7770–7772.

[50]E.Reading,etal.,Theroleofthedetergentmicelleinpreservingthestructure ofmembraneproteinsinthegasphase,Angew.Chem.Int.Ed.54(15)(2015) 4577–4581.

[51]B.L.Schwartz,K.J.Light-Wahl,R.D.Smith,Observationofnoncovalent complexestotheavidintetramerbyelectrosprayionizationmass spectrometry,J.Am.Soc.MassSpectrom.5(3)(1994)201–204.

[52]N.J.Martin,etal.,Nativeliquidextractionsurfaceanalysismassspectrometry: analysisofnoncovalentproteincomplexesdirectlyfromdriedsubstrates,J. Am.Soc.MassSpectrom.2015(2016)1–8.

[53]M.F.Bush,etal.,Collisioncrosssectionsofproteinsandtheircomplexes:a calibrationframeworkanddatabaseforgas-phasestructuralbiology,Anal. Chem.82(22)(2010)9557–9565.

(8)

[54]M.B.vanEldijk,etal.,EvidencethatthecatenaneformofCS2hydrolaseisnot anartefact,Chem.Commun.49(71)(2013)7770–7772.

[55]J.L.P.Benesch,etal.,Proteincomplexesinthegasphase:technologyfor structuralgenomicsandproteomics,Chem.Rev.107(8)(2007)3544–3567. [56]V.A.Mikhailov,etal.,Mass-selectivesoft-landingofproteinassemblieswith

controlledlandingenergies,Anal.Chem.86(16)(2014)8321–8328. [57]A.Dyachenko,etal.,Allostericmechanismscanbedistinguishedusing

structuralmassspectrometry,Proc.Natl.Acad.Sci.U.S.A.110(18)(2013) 7235–7239.

[58]E.Morag,E.A.Bayer,M.Wilchek,Immobilizednitro-avidinand

nitro-streptavidinasreusableafﬁnitymatricesforapplicationinavidin-biotin technology,Anal.Biochem.243(2)(1996)257–263.

[59]C.Brauckmann,etal.,Theinteractionofplatinum-baseddrugswithnative biologicallyrelevantproteins,Anal.Bioanal.Chem.405(6)(2013)1855–1864. [60]D.M.Mock,M.I.Malik,Distributionofbiotininhumanplasma:mostofthe