Method
Article
Immunoprecipitation
methods
to
identify
S-glutathionylation
in
target
proteins
Elena
Butturini
*
,
Diana
Boriero,
Alessandra
Carcereri
de
Prati,
So
fia
Mariotto
DepartmentofNeuroscience,BiomedicineandMovementSciences,SectionofBiologicalChemistry,University
ofVerona,Verona,Italy
*Correspondingauthor.
E-mailaddress:elena.butturini@univr.it(E.Butturini).
https://doi.org/10.1016/j.mex.2019.09.001
2215-0161/©2019TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
creativecommons.org/licenses/by-nc-nd/4.0/).
MethodsX6(2019)1992–1998
ContentslistsavailableatScienceDirect
MethodsX
A B S T R A C T
S-glutathionylationisareversiblepost-translationalmodificationofproteinsthatgenerateamixeddisulfide between glutathione to thiolate anion of cysteine residues in target proteins. In the last ten years, S-glutathionylationhasbeenextensivelystudiedsinceitrepresentsthecellularresponsetooxidativestress,in physiological aswell as pathological conditions. This modification may be a protective mechanism from irreversibleoxidativedamageand,ontheotherhand,maymodulateproteinfoldingandfunction.
DuetotheimportanceofS-glutathionylationincellularredoxsignaling,variousmethodshavebeendeveloped toidentifyS-gluthationylatedproteins.
Herein,wedescribetwoeasymethodstorecognizedS-glutathionylationofatargetproteinafteroxidative stressincellularextractsbasedondifferentimmunoprecipitationprocedures.Theimmunoprecipitationassay allowsthecaptureofoneglutathionylatedproteinusingaspecificantibodythatbindstothetargetprotein.The presenceofS-glutathionylationintheimmunoprecipitatedproteinisidentifiedusinganti-glutathioneantibody. Thesecondtypeofapproachisbasedonthedetectionoftheglutathionylatedproteinwithbiotin/streptavidin technique.Afterdifferentstepsofprotectionofnon-oxidizedthiolicgroupsandreductionofS-glutathionylated groups,thenewly-formedproteinfree-thiolsarelabeledwithbiotin-GSH.Themodifiedproteincanbeisolate withstreptavidin-beadsandrecognizedusinganantibodyagainsttargetprotein.
S-glutathionylationisareversiblepost-translationalmodificationofproteinsthatrecentlyhasbeenemerged asimportantsignalingintheredoxregulationofproteinfunction.
Bothmethodstoidentifyglutathionylatedproteinsareeconomic,easyanddonotrequireparticularequipment.
Thesetupsofbothmethodsguaranteehighreproducibility.
©2019TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A R T I C L E I N F O
Methodname:ImmunoprecipitationmethodstoidentifyS-glutathionylationintargetproteins
Keywords:Glutathionylation,Method,Immunoprecipitation,Bio-GEE
Articlehistory:Received21May2019;Accepted2September2019;Availableonline10September2019
SpecificationTable
SubjectArea: Biochemistry,GeneticsandMolecularBiology
Morespecificsubjectarea: Biochemistry
Methodname: ImmunoprecipitationmethodstoidentifyS-glutathionylationintargetproteins
Nameandreferenceof
originalmethod:
Jaffrey,S.R.,Snyder,S.H.,2001ThebiotinswitchmethodforthedetectionofS-nitrosylated
proteins.SciSTKE.2001(86):pl1
Resourceavailability: NA
Methoddetails Introduction
Glutathione (GSH) deficiency manifests itself largely through an increased susceptibility to oxidative stress whereas elevated GSH levels observed in many types of cancer cells, generally increase antioxidantcapacity.Oxidative stressmaycausereversible and/orirreversible oxidative modificationsofsensitiveproteinsthatmaymodulatetheiractivityorfunction.Mildoxidativestress inducesreversiblemodificationsthat protectproteinsfromirreversibledamageandmoduletheir function.Conversely,excessiveoxidativestresstriggersirreversiblemodificationsofthiolicgroupsof proteinsgenerallyassociatedwithpermanentlossoffunction,misfoldingandaggregationtendency [1].
Inmammaliancells,asignificantamountofGSHmaybereversiblyboundtoproteins,throughthe formation of mixed disulfides between GSH and protein sulfhydryl groups. The resulted
post-translationalmodification,knownasS-glutathionylation, notonlystoresGSH, preventingits loss underoxidativeconditions,butalsoprotectssensitiveproteinthiolsfromirreversibleoxidation[2]. It is important to note that protein S-glutathionylation occurs in pathological as well as physiologicalconditions,suggestingthepossibleinvolvementofthispost-translationalmodification incellularsignalingandredoxregulationofproteinfunction[1,2].
ProteinsulfhydrylsexhibitstrikingdifferentialsusceptibilitytoS-glutathionylation.Thus,whilea proteinmaycontainnumerouscysteineresidues,onlyaminorityofthesewillhavethechemical propertiestofunctionaspossibletargetsitesforoxidant.
Thetwomajorfactorsthatdeterminesusceptibilityofcysteinylresiduestoredoxreactionsarethe accessibilityof thethiolwithin thethree-dimensional structure of the proteinand thecysteine reactivity, which is influenced by the neighboring amino acids. Accessibility is defined by the secondaryandtertiarystructureoftheproteinanddependsontherelativeeasinessofthecysteineto beexposedtothesolvent.ThereactivityofSHgroupsismoredifficulttobepredicted.Thecysteine sulfhydrylsofmostcytoplasmicproteins havea pKavalueof8.5and,in thenormallyreducing environmentofthecytoplasmatphysiologicalpH,theseresiduesarealmostcompletelyprotonated. Asresult,theyareunlikelytobeS-glutathionylated.However,redox-sensitiveproteinshavespecific cysteineresidueslocalizedinabasicmicroenvironmentthatshifttheirpKatoalowervalue,nearor below7,atphysiologicalpH.ThispKavaluekeepsthecysteineinthethiolateformandmakesitan ‘activecysteine’,whichhasenhancedreactivityforGSH[3].
Herein,wedescribetwoeasymethodsdevelopedforidentifyingglutathionylatedproteinsthat could be used in parallel in order to exclude false positive results. The first one is the immunoprecipitationof targetproteinfollowedbyelectrophoresisundernon-reducingcondition andwesternblotanalysiswithanti-GSHantibody[4,5].Thismethodremainsthemostusedstrategy fortheidentificationofS-glutathionylatedproteins.Thesecondmethodisamodifiedbiotinswitch assayproposedasdescribedpreviouslybyJaffreyandSnyder[6]withmodifications.Thismethodis basedontheuseofbiotinylatedglutathionethatcanreactwithsensitiveredoxthiolsgroupofcysteine residues.Themodifiedproteincanbeisolatebyaffinitycapturewithstreptavidin-conjugatedmedia andrecognizedbywesternblot[7,8].
Themethodsproposedinthismanuscriptwereusedbythesameauthorsintwopreviouspapersin whichthelevelofS-glutathionylationofSTAT3andSTAT1wereanalysedincellsafteroxidativestress. Toidentifythemodifiedcysteineresidue,oxidizedSTAT1andSTAT3werealsoanalysedbymass spectroscopy.Theobtaineddataconfirmedthereliabilityoftheproposedmethods[7–9].
Materials Buffers
1 RIPAlysisbuffer:50mMTris HClpH7.5,150mM NaCl,1%(v/v) Igepal(CA-630I3021, Sigma-Aldrich), 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate (SDS), 1mM ethylenediaminetetraaceticacid(EDTA),10%(v/v)glycerol,100mMNaF,1mMNa3VO4.
2 Samplebuffer:62.5mMTris HClpH6.8, 10%(v/v)glycerol,5%(w/v)SDS,0.05%(w/v)bromophenol blue,5%(v/v)β mercaptoethanol.
3 Non-reducingSamplebuffer:samplebufferwithoutβ mercaptoethanol. 4 Runningbuffer:25mMTris HClpH8.3,190mMglycine,0.1%(w/v)SDS. 5 Transferbuffer:25mMTris HClpH8.3,192mMglycine,20%(v/v)methanol. 6 TBSbuffer:20mMTris HClpH7.5,150mMNaCl.
7 Washingbuffer:TBSbuffercontaining0.1%(v/v)Tween-20detergent 8 Blocking:washingbuffercontaining3%(w/v)BSA.
9 Strippingbuffer:62.5mMTris HClpH6.7,2%(w/v)SDS,100mMβ mercaptoethanol.
Biotinylatedglutathione(BioGSH)synthesis
Dissolvethewater-solublesulfosuccinimidyl-6-(biotinamido)-hexanoate(sulfo-NHS-biotin)and GSH1:1inwaterandadjustthepHto7.2withNaOH.Incubatethereactionatroomtemperaturefor 1h.Unreactedbiotinreagentisquenchedbytheadditionof1MTris HClpH7.2.
Reagents
1 N-ethylmaleimide(NEM)(E3876,Sigma-Aldrich):Prepare250mMNEMinwater.Thissolutionis stableforatleast3monthsat 20C.
2 1,4-Dithiothreitol(DTT)(Sigma-Aldrich):freshlyprepare1MDTTinwateranddonotstore. 3 Diamide(D3648,Sigma-Aldrich):prepare100mMdiamideinwater.Thissolutionisstableforat
least3monthsat 20C.
4 ProteinAorproteinGsepharose:preparetheresinfollowingthemanufacturer’sinstructions,mix thoroughlyandequilibratewithRIPAbuffer.Theslurryisnowreadyforuse.Itcanbestoredat4C
forafewdays.IfusingamonoclonalantibodychooseproteinG-coupledsepharosebeads,ifusinga polyclonalantibodyproteinA-coupledsepharosebeadsareusuallysuitable.
5 Streptavidin-agarosebeads (ThermoFisherScientific):prepare the beads followingthe manu-facturer’sinstructions,mixthoroughlyandequilibratewithwashingbuffer.
6 Agarose beads (ThermoFisher Scientific): prepare the beads following the manufacturer’s instructions,mixthoroughlyandequilibratewithwashingbuffer.
7 Chemiluminescencekit(ImmobilonWestern,Millipore).
8 Anti-Glutathionemonoclonalantibody(ViroGen):diluteantibody1:1000 inblockingbuffer. 9 Anti-mouse IgG peroxidase-conjugated antibody (Cell Signaling Technology): dilute antibody
1:2000inblockingbuffer.
10Lowryreagent(Sigma-Aldrich)andBSAstandardcurve.
Immunoprecipitationmethod 1 Seed5105
–1106 cells inculture dish and culturethem until con
fluence.Treat cells with appropriateoxidant.Forexample,1mMdiamidefor30min.Nottreatedcellsareusedascontrol. 2 Placeculturedishesoniceandwashthecellswithice-coldPBS.Addnewice-coldPBSandscrape adherentcellsoffthedishusingacoldplasticcellscraperthengentlytransferthecellsuspension intoapre-cooledmicrocentrifuge.Centrifugesamplesat4Canddiscardthesupernatants. 3 LysecellsinRIPAbuffersupplementedwith50mMNEMandwithproteaseinhibitors(10
m
g/mLantipain,5
m
g/mLpepstatin,1mMphenylmethylsulfonylfluoride)for30minonice. 4 Centrifugesamplesat14,000gfor30minat4Ctopelletthecelldebris.5 GentlytransfersupernatantstonewEppendorftubes.
6 QuantifytotalproteinscontentusingLowryreagentandBSAstandardcurve.
7 Clarify1mgofproteinsfromcelllysatesusing40
m
LofslurryofproteinA-orG-sepharosein Eppendorftubes.Incubateat4Cfor1hundergentlerotation.Pre-clearingthelysatecanhelpto reducenon-specificbindingandreducebackground.8 Centrifugesamplesat1000gat4Cfor1–2min. 9 GentlytransfersupernatantstonewEppendorftubes.
10Incubateequalamountsofproteinsfromtheclarifiedcelllysateswiththeantibodyagainstthetarget proteinat4Cfor12h,preferablyundergentlerotation.Thelengthoftheincubation perioddependson theamount ofproteinandaffinitypropertiesof theantibody. Checkthe antibodydatasheetfor recommendedantibodyconcentration.Asaguidelineuse1
m
gofaffinitypurifiedpolyclonalantibody. 11 Centrifugesamplesat1000gat4Cfor1–2min.12Add60
m
LofslurryofproteinAorGsepharoseandmixat4Cundergentlerotationfor2h.13Centrifugesamplesat1000gat4Cfor1–2min.
14Collectthebeadsanddiscardthesupernatants.Add200
m
LofRIPAbuffertothetubewiththe beadsandgentlymix.Repeatthiswashtwice.15Add30
m
Lof2Xnon-reducingSamplebuffertothecollectedbeadsandheatthesamplesat100C for3min.16Centrifugesamplesat1000gat4Cfor1–2minandcollectthesupernatantsforwesternblot analysis.
17 SeparateProteinAorGpull-downsamplesona5–10%SDS–polyacrylamidegelinrunningbuffer followingthemanufacturer’sinstructionsoftheelectrophoresisapparatus.
Transfer proteins to PVDF membrane using transfer buffer following the manufacturer’s instructionsofthetransferapparatus.
19Blocknon-specificbindingonthemembranebyincubationinblockingbufferatroomtemperature for1hundergentleagitation.
20ProbemembranewithmonoclonalantibodyagainstGSHat4Covernightundergentleagitation.
21Washthemembraneinwashingbufferfor5minundergentleagitation.Repeatwashthreetimes. 22Incubateblotswithanti-mouseIgGperoxidase-conjugatedantibodyatroomtemperaturefor1h
undergentleagitation.
23Washthemembraneinwashingbufferfor5minundergentleagitation.Repeatwashthreetimes. 24Detect protein-antibody reactions with chemiluminescent detection reagent following the
manufacturer’sinstructions.Acquireimageswithanautomatedimageacquisitionsystem. Tochecktheimmunoprecipitatedprotein,removeprimaryandsecondaryantibodiesfromthe membraneandre-probeitwiththeprimaryantibodyagainstthetargetedprotein.
25Incubatemembraneinstrippingbufferat50Cfor30minundergentleagitation.
26Checktheefficiencyofstrippingbyincubatingthemembranewithchemiluminescentdetection reagent.
27
Fig.1.Modifiedbiotinswitchassay.
Ifstrippingisjudgedtobesatisfactory,rinsethemembraneseveraltimeswithwashingbuffer, thenblockwithblockingbuffer,1hundergentleagitation.
28 Probethe membranewith antibodyagainst targetprotein.Check the antibodydatasheet for recommendedantibodyconcentration.
29 Repeatstep19–22forthedetectionoftheprotein.
Modifiedbiotinswitchassaymethod
1 CellsarelysedinRIPAbuffersupplementedwithproteaseinhibitors(10
m
g/mLantipain,5m
g/mL pepstatin,1mMphenylmethylsulfonylfluoride)onicefor30min.2 Centrifugesamplesat14,000gat4Cfor30mintopelletthecelldebris. 3 TransfersupernatantstonewEppendorftubes.
4 QuantifytotalproteinscontentusingLowryreagentandBSAstandardcurve.
5 Incubate1mgofproteinsfromcelllysateswith1mMdiamideorotheroxidizingagentonicefor 30min.Forthecontrolsample,incubate1mgofproteinsfromcelllysateswithoutoxidantagents andfollowthesameprocedure.
6 Transfer the samples in Amicon1 Ultra spin desalting column (Millipore) and follow the manufacturer’sinstructionstoremovecellularGSHandtheoxidantsinexcess.
7 TransferthecollecteddesaltedsamplesinnewEppendorftubes.
8 Add50mMNEMandkeeponicefor20mintostablyalkylatethefreethiols.
9 ReducethethiolgroupsthatarenotalkylatedbyNEMwith60mMDTTkeepingthesolutiononice for20min.
10Transferthe samplesin Amicon1 Ultra columnfollowing the manufacturer’s instructions to removefreeNEMandDTT.
11 TransfereachsampleinnewEppendorftube.
12Oxidizeagainthefreethiolgroupswith1mMdiamideorotheroxidizingagentsonicefor30min. 13Incubatethesampleswith1mMBioGSHonicefor30min.Otherwise,thecommerciallyavailable BiotinylatedGlutathioneethyleneester(BioGEE,MolecularProbes,ThermoFisherScientific)can beusedtolabeltheredoxsensitivecysteine.
14TransferthesamplesinAmicon1 Ultracolumnand followthemanufacturer’sinstructions in ordertoremovecellularGSHandtheoxidantsinexcess.
15Replacethebufferwith400
m
LcoldRIPAbuffersupplementedwithproteaseinhibitors(10m
g/mL antipain,5m
g/mLpepstatin,1mMphenylmethylsulfonylfluoride)andtransfereachsamplein newEppendorftube.16QuantifyproteinscontentusingLowryreagentandBSAstandardcurve
17 Incubate1mgofproteinswith100
m
LofslurryagarosebeadsinEppendorftubesat4Cfor1h under gentle rotation. This pre-clearing step can help to reduce non-specific binding and background.18 Centrifugesamplesat1000gat4Cfor1–2min.
19TransfersupernatantstonewEppendorftubesandadd100
m
Lofstreptavidin-agarosebeads.Mix underrotationat4Cfor2h.20 Centrifugesamplesat1000gat4Cfor1–2min.
21Collectthebeadsanddiscardthesupernatants.Add200
m
LofRIPAbuffertothetubewiththe beads and gently mix. Centrifuge samples at 1000g at 4C for 1–2minand discardthe supernatants.Repeatthiswashtwice.22 Add30
m
Lof2XSamplebuffertothecollectedbeadsandheatthesamplesat100Cfor3min.23 Centrifugesamplesat1000gat4Cfor1–2minandcollectthesupernatantsforSDS-PAGEand WesternBlotanalysis.
24 Separate Streptavidinpull-down samples on a 5–10% SDS–polyacrylamide gel (SDS-PAGE) in runningbufferfollowingthemanufacturer’sinstructionsoftheelectrophoresisapparatus. 25 Transfer proteins to PVDF membrane using transfer buffer following the manufacturer’s
instructionsofthetransferapparatus. 26
Blocknon-specificbindingonthemembranebyincubationinblockingbufferatroomtemperature for1hundergentleagitation.
27Probethemembranewithprimaryantibodyagainsttargetproteinovernightat4Cundergentle agitationusingtherecommendeddilutioninblockingsolution.Checktheantibodydatasheetfor recommendedantibodydilution.
28Washthemembraneinwashingbufferfor5minundergentleagitation.Repeatwashthreetimes. 29Incubate the membrane with secondary antibody peroxidase-conjugated for 1h at room temperatureundergentleagitationusingtherecommendeddilutioninblockingsolution.Check theantibodydatasheetforrecommendedantibodydilution.
30Washthemembraneinwashingbufferfor5minundergentleagitation.Repeatwashthreetimes. 31Detect protein-antibody reactions with chemiluminescent detection reagent following the
manufacturer’sinstructions.Acquireimageswithanautomatedimageacquisitionsystem. Aworkflowof representationof modifiedbiotin switchprocedure fortheidentificationof S-glutathionylatedproteinisreportedinFig.1.
References
[1]D.Giustarini,R.Rossi,A.Milzani,R.Colombo,I.Dalle-Donne,S-glutathionylation:fromredoxregulationofproteinfunctions tohumandiseases,J.Cell.Mol.Med.8(2)(2004)201–212.
[2]I.Dalle-Donne,R.Rossi,G.Colombo,D.Giustarini,A.Milzani,ProteinS-glutathionylation:aregulatorydevicefrombacteria tohumans,TrendsBiochem.Sci.34(2)(2009)85–96.
[3]S.G.Rhee,Y.S.Bae,S.R.Lee,J.Kwon,Hydrogenperoxide:akeymessengerthatmodulatesproteinphosphorylationthrough cysteineoxidation,Sci.STKE2000(53)(2000)pe1.
[4]E.Butturini,E.Cavalieri,A.C.dePrati,E.Darra,A.Rigo,K.Shoji,N.Murayama,H.Yamazaki,Y.Watanabe,H.Suzuki,S. Mariotto,Twonaturallyoccurringterpenes,dehydrocostuslactoneandcostunolide,decreaseintracellularGSHcontentand inhibitSTAT3activation,PLoSOne6(5)(2011)e20174.
[5]E.Butturini,A.CarcereridePrati,G.Chiavegato,A.Rigo,E.Cavalieri,E.Darra,S.Mariotto,Mildoxidativestressinduces S-glutathionylationofSTAT3 andenhanceschemosensitivityoftumouralcellstochemotherapeuticdrugs,FreeRadic.Biol. Med.65(2013)1322–1330.
[6]S.R.Jaffrey,S.H.Snyder,ThebiotinswitchmethodforthedetectionofS-nitrosylatedproteins,Sci.STKE2001(86)(2001)pl1. [7]E.Butturini,E.Darra,G.Chiavegato,B.Cellini,F.Cozzolino,M.Monti,P.Pucci,D.Dell’Orco,S.Mariotto,S-Glutathionylationat
Cys328andCys542impairsSTAT3phosphorylation,ACSChem.Biol.9(8)(2014)1885–1893.
[8]E.Butturini,F.Cozzolino,D.Boriero,A.CarcereridePrati,M.Monti,M.Rossin,D.Canetti,B.Cellini,P.Pucci,S.Mariotto, S-glutathionylationexertsopposingrolesintheregulationofSTAT1andSTAT3signalinginreactivemicroglia,FreeRadic.Biol. Med.117(2018)191–201.
[9]E.Butturini,D.Boriero,A.CarcereridePrati,S.Mariotto,STAT1drivesM1microgliaactivationandneuroinflammationunder hypoxia,Arch.Biochem.Biophys.669(2019)22–30,doi:http://dx.doi.org/10.1016/j.abb.2019.05.011.
