Patterns of Protein Synthesis and Tolerance of Anoxia in Root

ProteinSynthesisandToleranceofAnoxia

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(1996).Intactseedlingswerefirsttreatedinglassfunnels,asdescribedabove.Priortoanoxia,seedlingsweretrans-ferredintoasealedNMRsampletube,andspectrawereobtainedat202.5MHzonaspectrometer(modelGN500,GeneralElectric,Fairfield,CT).Gasesequilibratedwith0.1mmCaSO4wereusedforperfusionwithaconstantgasstreamthroughthesampletubeduringtheexperiment.CytoplasmicpHwasestimatedfromthechemicalshiftsofcytoplasmicPi(Roberts,1986).

ProteinIdentificationbyMS

Individualorpooledgelspots(2–3spots)fromseparateCoomassieBlue-stainedorsilver-stainedtwo-dimensionalgels(100 gprotein/gel)weresubjectedtotrypticdiges-tionusingamodifiedprocedureofRosenfeldetal.(1992).Gelspotscontainingproteinswereexcisedfromgelsusingascalpelinalaminarflowhood.Theexcisedgelspotswerestoredin100 LofHPLC-gradewaterat4°Cuntilsubse-quentanalyses.Thespotswerethenmincedandwashedwith25mmNH4HCO3in50%(v/v)acetonitrile.Thegelpieceswereallowedtodryandthenrehydratedin25mmNH4HCO3with0.5to1.0 goftrypsinat37°Covernight.Afterdigestion,thedigestionsolutionwasseparatedfromthegelslices,andthegelsliceswerewashedwithHPLC-gradewateronceandwith50%(v/v)acetonitrile,5%(v/v)trifluoroaceticacidthreetimesatroomtemperaturetoextractthepeptidesfurther.Pooledextracts(includingthedigestionsolutionandboththeaqueousandorganicwashes)wereconcentratedusingaSpeed-Vac(SavantIn-struments,Holbrook,NY).Insomecases,sampleswerefurtherfractionatedbyreversedphaseHPLConamicro-boreC18column(1.0mm 15cm;Vydac,TheSeparationsGroup,Hesperia,CA).HPLCfractionswerecollectedandconcentrated.

Trypticpeptidemassesweremeasuredbyanalyzingone-twentiethofeachconcentratedsampleafterdigestion(orone-tenthofeachHPLCfraction)usingamatrix-assistedlaserdesorption-ionizationdelayedextractionreflectrontime-of-flight(MALDI-DE-TOF)massspec-trometerequippedwithanitrogenlaser( 337nm)(Voyager-DESTR,PEBiosystems,Framingham,MA).Peptideswereco-crystallized1:1(v/v)withmatricescon-sistingofsaturated -cyano-4-hydroxycinnamicacidpre-paredin50%(v/v)acetonitrile/1%(v/v)trifluoroaceticacid.AllMALDIspectrawereeitherexternallycalibratedusingastandardpeptidemixtureorinternallycalibratedusingtrypsinauto-proteolysisproducts.Mono-isotopicmassesfromallspectrarecordedforagivenpeptidearereported.Forseveralpeptidesthatexhibitedthehighestpseudo-molecularionabundanceonMALDImassspectra,partialaminoacidsequencewasdeterminedusingpost-sourcedecayanalysis.

Matchingofexperimentalresults(measuredpeptidemassvalues)withtheoreticaldigestsandsequenceinfor-mationobtainedfromvariousdatabaseswasperformedusingtwosequencedatabasesearchprograms,MS-Fitand

MS-Tag(Jimenezetal.,1998;Clauseretal.,1999).TheseprogramsweredevelopedbyKarlClauserandPeterBakeroftheNationalInstitutesofHealth(NIH)/NationalScienceFoundationMassSpectrometryFacility,UniversityofCalifornia,SanFrancisco,andareavailableathttp://pros-pector.ucsf.edu/.MS-Fitallowstheusertomatchtheob-servedtrypticpeptidemassesofanunknownproteintotheexpectedpeptidemassesofanyproteinforwhichaminoacidornucleotidesequenceinformationisavailable.Data-basequerieswerecarriedoutformono-isotopicpeptidemassesusingthefollowingparameters:peptidemasstol-eranceof 50ppm(ppm [experimentalmass(indal-tons) theoreticalmass]/theoreticalmass,expressedinpartspermillion),equivalentto0.1Dfora2-kDpeptide;themaximumnumberofmissedtrypticcleavagesof2or3;andmodificationsincludingconversionofpeptideN-terminalGlntopyro-Gln,oxidationofMet,acetylationoftheNterminus,andmodificationofCysbyacrylamide.DatabasesearchesusingMS-Tagtomatchpost-sourcedecay(PSD)fragmentions(alongwiththemassofapre-cursorion)usedthefollowingparameters:precursorionmasstoleranceof 100ppm(measuredbyMALDI-MS)andPSDfragmentionmasstoleranceof 1,500ppm.Databasessearchedincludedproteindatabasessuchasthenon-redundantNCBInrcompiledbytheNational

Center

Figure1.Effectofdurationofhypoxicpretreatmentonmaizeroottiptoleranceto13hofanoxia.Intactseedlingswerepretreatedunderhypoxiaforvariouslengthsoftime,followedby13hofanoxiaand26hofnormoxia(seeschematic,“Experimentalplan”).Tolerancewasassessedusingrootgrowthandroottipviabilityassays.Growthdataaremeanvalues SE(n 10).Viabilitydataareaggregatesofthreeindependentexperiments,fromobservationsofatotalof30seedlingsforeachpoint.Normoxiccontrolseedlingsofthesamedevelopmentalagewereexposedto100%(v/v)O2only.