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NEB/Protein Deglycosylation Mix II/P6044S/20 reactions
  • NEB/Protein Deglycosylation Mix II/P6044S/20 reactions

NEB/Protein Deglycosylation Mix II/P6044S/20 reactions

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    • Description:

      Glycosylationisoneofthemostcommonpost-translationalmodificationsofproteins,asshowninFigure1.N-linkedglycosylationoccurswhenglycansareattachedtoasparagineresiduesonthecoreprotein.O-linkedglycosylationoccurswhenglycansareattachedtoserineorthreonineresidues.Bothchemicalandenzymaticmethodsexistforremovingoligosaccharidesfromglycoproteins.However,chemicalmethodssuchasβ-eliminationwithmildalkaliormildhydrazinolysiscanbeharshandmayresultinincompletesugarremovalanddegradationoftheprotein;whereas,enzymaticmethodsaremuchgentlerandcanprovidecompletesugarremovalwithnoproteindegradation.

      PNGaseFisthemosteffectiveenzymaticmethodforremovingalmostallN-linkedoligosaccharidesfromglycoproteins.PNGaseFdigestiondeaminatestheaspargineresiduetoasparticacid,andleavestheoligosaccharideintact,keepingitsuitableforfurtheranalysis.Oligosaccharidescontainingafucoseα(1-3)-linkedtotheglycancoreare,however,resistanttoPNGaseFwhichcanoccuronsomeplantandinsectglycoproteins.

      ToremoveO-linkedglycans,monosaccharidesmustberemovedbyaseriesofexoglycosidasesuntilonlytheGalβ1-3GalNAc(core1)and/ortheGlcNAcβ1-3GalNAc(core3)coresremainattachedtotheserineorthreonine.NEB’sO-Glycosidase,clonedfromEnterococcusfaecalis,canthenremovethesecorestructureswithnomodificationoftheserineorthreonineresidues.Anymodificationofthecorestructures,includingsialyation,willblocktheactionoftheO-Glycosidase.Sialicacidresiduesareeasilyremovedbyageneralα2-3,6,8,9NeuraminidaseA.Inaddition,exoglycosidasessuchasβ(1-4)GalactosidaseSandβ-N-Acetylhexosaminidasefcanbeincludedindeglycosylationreactionstoremoveothercomplexmodificationsoftenknowntobepresentonthecorestructures.ThiscombinationofenzymesmaynotremoveallO-linkedoligosaccharidesbutshouldremovemanycommonoligosaccharidestructures.

      TheProteinDeglycosylationMixIIcontainsalloftheenzymes,reagents,andcontrolsneededtoremoveallN-linkedandsimpleO-linkedglycansaswellassomecomplexO-linkedglycans.Thismixcontainsenzymesufficientfor20reactionsorthecleavageofasmuchas2mgofglycoprotein.AlloftheenzymesandreagentsincludedintheProteinDeglycosyationMixIIareMassSpectrometrycompatIBLe.Followingthedeglycosylationreaction,samplesarereadytobepreparedformassspectrometryanalysis.

      Figure1:AGlycoproteinmodifiedwithO-linkedandN-linkedglycosylation.

      P6044_figure_1
      Figure2


      EnzymaticDeglycosylationofBovineFetuinunderbothnative(10XDeglycosylationMixBuffer1)andreducing(10XDeglycosylationMixBuffer2)conditions.20μgreactionswereloadedontoa10-20%Tris-glycineSDS-PAGEgel.
      Lane1:???ColorPrestainedProteinStandard,BroadRange(11-245?kDa)(NEB#P7712)
      Lane2:???20?μguntreatedFetuincontrol
      Lane3:???20μgFetuindeglycosylatedundernativeconditionswithDeglycosylationMixBuffer1
      Lane4:???20μgFetuindeglycosylatedunderreducingconditionswithDeglycosylationMixBuffer2
      Lane5:???5μlProteinDeglycosylationMixII.

      ProteinDeglycosylationMixII:
      PNGaseF(Glycerol-free),Recombinant:
      10,000units/vial

      O-Glycosidase:
      80,000units/vial

      α2-3,6,8,9NeuraminidaseA:
      400units/vial

      β1-4GalactosidaseS:
      960units/vial

      β-N-acetylhexosaminidasef:
      300units/vial

      SubstrateControl:
      Fetuin,0.5mg(FetuincontainssialylatedN-linkedandO-linkedglycans)

      ofEnzymesIncludedintheProteinDeglycosylationMixII
      O-Glycosidase(NEB#P0733),alsoknownasEndo-α-N-Acetylgalactosaminidase,isarecombinantenzymeclonedfromEnterococcusfaecalis(1).Itcatalyzestheremovalofcore1andcore3O-linkeddisaccharidesfromglycoproteins.Themolecularweightisapproximately147kDa.

      PNGaseF(Glycerol-free),Recombinant(NEB#P0709),alsoknownasPeptide:N-glycosidaseF,isclonedfromElizabethkingiamiricola(formerlyFlavobacteriummeningosepticum)andexpressedinE.coli(2).PNGaseF(Glycerol-free),RecombinantisanamidasewhichcleavesbetweentheinnermostGlcNAcandasparagineresiduesofhighmannose,hybrid,andcomplexoligosaccharidesfromN-linkedglycoproteinsunlessα(1-3)corefucosylated.Themolecularweightisapproximately36kDa.

      α2-3,6,8,9NeuraminidaseA(NEB#P0722),alsoknownasSialidaseA,isarecombinantenzymeclonedfromArthrobacterureafaciensandexpressedinE.coli(3).Itcatalyzesthehydrolysisofα2,3,α2,6,α2,8andα2,9linkedN-acetylneuraminicacidresiduesfromglycoproteinsandoligosaccharides.Themolecularweightisapproximately100kDa.

      β1-4GalactosidaseS(NEB#P0745),isarecombinantenzymeclonedfromStreptococcuspneumoniaeandexpressedinE.coli(4).Itisahighlyspecificexoglycosidasethatcatalyzesthehydrolysisofβ1-4linkedgalactoseresiduesfromoligosaccharides.Themolecularweightisapproximately231kDa.

      β-N-Acetylhexosaminidasef(NEB#P0721),isarecombinantenzymeclonedfromStreptomycesplicatus(5)andoverexpressedinE.coli(6).Itcatalyzesthehydrolysisofterminalβ-N-acetylgalactosamineandglucosamineresiduesfromoligosaccharides.Themolecularweightisapproximately100kDa.

      ReagentsSupplied

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      DeglycosylationMixBuffer1-2010X
      DeglycosylationMixBuffer2-2010X

      Notes:

      TheProteinDeglycosylationMixIIissuppliedwithtwodifferentreactionbuffers.Pleasereferencethe“ProtocolsandManuals”tabforthespecificprotocolassociatedwitheachbuffersystem.The10XDeglycosylationMixBuffer1(NEB#B6044)shouldbeusedwhennative(non-denaturing)conditionsarenecessary.The10XDeglycosylationMixBuffer2(NEB#B6045)willreducetheglycoprotein,butwillalsoprovidethemostefficientandcompletelevelofdeglycosylation.Ifnon-denaturingconditionsarenotrequired,werecommendusing10XDeglycosylationMixBuffer2.Storage:Itisrecommendedtostorethiskitat4°C.Allcomponentsofthekitwillbestableforatleastoneyearifstoredcorrectly.TheProteinDeglycosylationMixIIisnotrecommendedforuseonMucin-likesubstrates.
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