Extracellular Matrix Proteome and Phosphoproteome of Potato

22 Pages·2016·1.93 MB·English

proteomes
Article
Extracellular Matrix Proteome and Phosphoproteome
of Potato Reveals Functionally Distinct and Diverse
Canonical and Non-Canonical Proteoforms
EmanElagamey†,KanikaNarula,ArunimaSinha,PoojaRaniAggarwal,SudipGhosh,
NiranjanChakrabortyandSubhraChakraborty*
NationalInstituteofPlantGenomeResearch,ArunaAsafAliMarg,NewDelhi110067,India;
[email protected](E.E.);[email protected](K.N.);[email protected](A.S.);
[email protected](P.R.A.);[email protected](S.G.);[email protected](N.C.)
* Correspondence:[email protected]@nipgr.ac.in;Tel.:[phone]
† PresentAddress:AgriculturalResearchCenter(ARC),PlantPathologyResearchInstitute,9Gamaast.,
Giza12619,Egypt.
AcademicEditors:NicolasL.TaylorandJacekR.Wisniewski
Received:27March2016;Accepted:13June2016;Published:24June2016
Abstract: The extracellular matrix (ECM) has a molecular machinery composed of diverse
proteinsandproteoformsthatcombinepropertiesoftensilestrengthwithextensibilityexhibiting
growth-regulatory functions and self- and non-self-recognition. The identification of ECM
proteoforms is the prerequisite towards a comprehensive understanding of biological functions
accomplished by the outermost layer of the cell. Regulatory mechanisms of protein functions
relyonpost-translationalmodifications,phosphorylationinparticular,affectingenzymaticactivity,
interaction,localizationandstability. ToinvestigatetheECMproteoforms,wehaveisolatedthecell
wallproteomeandphosphoproteomeofatuberouscrop,potato(Solanumtuberosum). LC-MS/MS
analysis led to the identification of 38 proteins and 35 phosphoproteins of known and unknown
functions. The findings may provide a better understanding of biochemical machinery and the
integratedproteinandphosphoproteinnetworkofECMforfuturefunctionalstudiesofdifferent
developmentalpathwaysandguidancecuesinmechanosensingandintegritysignaling.
Keywords: ECM;proteoforms;phosphoproteome;potato;2-DE;proteininteractionnetwork
1. Introduction
Cellular homeostasis, semantics, and mechanical attributes are complex processes involving
recruitment of cellular fibrils and differentiation of extracellular matrix (ECM). The extracellular
matrixexhibitsaprominentfeatureofcombiningextremetensilestrengthwithextensibility. Plasticity
of ECM is a prerequisite for the expansion and elongation of the cell by helping reorganize cell
wall networks and remodel cellular machinery. Cellular growth depends on the balance between
mechanicalforceandturgorpressuremechanics.Thisrepresentsanexusofvariousbiologicalprocesses
linkinggrowthanddevelopment,plant-pathogeninteractions,abioticstress,self-andinter-organismal
recognition, signaling components and metabolic processes. ECM integrates various internal and
external cues, notably nutritional, stress factors, and auto-regulatory signals. Composition and
structureofECMvariesacrossplantkingdom. Cellmorphogenesisisgovernedbythefunctional
integrity and maintenance mechanism in the ECM [1]. Therefore, assembly and disassembly of
ECMcomponentsormodulationofsignaltransductionpathwaysintermsofbiologicalactivityis
acommontheme.
ECMhydration,deposition,extension,cross-linkingandmechanosensingareaccuratelyregulated
inatemporal,spatial,anddevelopmentalmannerattranslationalandpost-translationallevels. The
Proteomes2016,4,20;doi:10.3390/proteomes4030020 www.mdpi.com/journal/proteomes
Proteomes2016,4,20 2of22
multifunctional feature of ECM houses a compendium of dynamic, heterogeneous, and complex
proteinsandphosphoproteins. ECMproteinscompriselessthan10%ofcellwalldryweight[2]and
representacomprehensivecatalogofthecellwallproteinsthatspreadthroughouttheextracellular
milieu and range from highly mobile with no apparent interaction, to covalently-bound proteins.
ECMproteinpopulationsofmanyplantorgansandtissueshavebeenreported[3–7]togetherwith
detailedmethodologiestooptimizeextraction[8–10]. Infact, differentECMproteins, particularly
phosphoproteinepitopes,arenotevenlydistributed(oraccessible)withinthecellwall,norarethese
epitopes distributed uniformly across two adjacent walls [11]. Reversible and sub-stoichiometric
proteinphosphorylationisanimportantposttranslationalmodificationforcell-cellcommunication
andsignaltransductionincellularprocesses. However,phosphorylationstudiesofECMproteinsin
plantshavenotbeeninvestigatedtodate. Phosphoproteomicsthatexaminesphosphorylationchanges
mayhelpindeterminingECMsignalingregulationatsystemlevel.
Potato (Solanum tuberosum L.) is the fourth most important temperate food crop consumed
worldwide and has about 200 wild species of various ploidy levels. Of which, tetraploids are
the most commonly cultivated species of potato all over the world. It is an excellent example
of a vegetatively-propagated plant species with complex genetics/genome [12]. There are many
unansweredquestionsofhowcellwalldynamicsareregulatedandcoordinatedwithothercellular
processes in potato. Proteoform subsets in different organelles fulfil discrete, but varied, cellular
functions and provide additional important information about protein localization and pathway
compartmentalization. Thus,understandingECMproteoformsmightplaymajorroleinunraveling
translationalandpost-translationalinformationthatmodulatesthebiologicalprocessesandmolecular
functionsintheoutermostlayerinpotato.
Here,weperformedapreliminaryproteomicandphosphoproteomicanalysisoftheextracellular
matrixoftetraploidpotatousingtwo-dimensionalgelelectrophoresis(2-DE)incombinationwith
LC-MS/MSanalyses. Functionalcataloguingofproteinsandphosphoproteinsfrompurifiedpotato
ECMfractiondefinedtranslationalactivitiesandarchitecturalreorganizationdynamics. Thisstudy
presentsthefirstECMproteomeandphosphoproteomereportinatubercrop. Furthermore,protein
andphosphoproteinnetworkhighlightsthatwall-associatedsignalingcomponents,wallhydration,
extension,deposition,andmechonosensingarethemajorfunctionsofECMinplantcells.
2. ExperimentalSection
2.1. PlantMaterialandGrowthCondition
Potato(SolanumtuberosumL.)cultivarKufriChipsona-1wasgrownandsubculturedonMurashige
and Skoog MS media [13] by taking seven node explants to get a uniform size of seedlings in
anenvironmentally-controlledgrowthroommaintainedat25˝C˘2˝C,50%˘5%relativehumidity,
under a 16 h photoperiod (270 µmol¨m´2¨s´1 light intensity). The three-week-old seedlings were
sampledasexperimentalmaterials,harvested,frozeninliquidnitrogenandstoredat´80˝C.
2.2. IsolationofPureECM
The ECM fraction was isolated as described by Averyhart-Fullard et al., and Feiz et al. with
fewmodifications[8,14]. Further,theECMfractionwaspurifiedasdescribedearlier[15]. Inbrief,
5.0goftissuesweregroundtopowderinliquidnitrogenwith0.3%(w/w)polyvinylpolypyrollidone
(PVPP)andtransferredto50mLcentrifugetube. Immediately,tissuepowderwashomogenizedin
lowionicstrengthhomogenizingbuffer(5mMK PO ,pH6.0,5mMDTT,1mMPMSF)for2minto
3 4
preservetheionicbondsandtodilutetheionicstrengthoftheECM.TheECMfractionwasrecovered
bydifferentialcentrifugationat1000ˆgfor5minat4˝C.Thepelletthusobtainedwaswashedten
timeswithexcessdeionizedwater.
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2.3. LightMicroscopy
ThepurifiedECMfractionwasvisualizedusinglightmicroscopyasdescribedinBessireetal.[16].
Inbrief,thepurifiedECMfractionwasfixedwithKarnovsky’sfixative(2%(v/v)paraformaldehyde
and 2.5% (v/v) glutaraldehyde) overnight at 4 ˝C. It was then washed in 0.1 M phosphate buffer
pH7.4andtreatedwith1%osmiumtetroxide. Thefractionwasdehydratedsequentiallyinacetone
andembeddedinepoxyembeddingresin(453591EA-F;SigmaAldrich,St. Louis,MO,USA).Thin
sectionsweremadeandstainedwithtoluidineblueandexaminedbyaNikonEclipse80imicroscope
(Nikon,Minato,Tokyo,Japan)
2.4. ECMProteinExtractionandQuantification
TheECMfractionwassuspendedinthreevolumes(w/v)ofextractionbuffer(200mMCaCl ,
2
5mMDTT,1mMPMSF,0.3%(w/w)PVPP)andextractedonashakingplatformfor2hat4˝Cto
remove any further contaminants and to release ECM proteins. Proteins were separated from the
insolubleECMfractionbycentrifugation(10,000ˆg)for10minat4˝Candfilteredthrougha0.45µm
filter. ThefiltratewasconcentratedusingaCentriconYM3(Millipore,Bedford,MA,USA)andthen
dialyzedovernightagainst1000volumesofdeionizedwaterwithonechange. Theconcentrationof
proteinextractwasdeterminedbyBradfordassay(Bio-Rad,Hercules,CA,USA).
2.5. IsolationofPlasmaMembraneProteins
ExtractionofplasmamembranefractionwascarriedoutaccordingtoSantonietal.[17]. Fifteen
gofthree-week-oldseedlingswerehomogenizedinhomogenizationbuffer(50mMsucrose,50mM
tris,10%(w/v)glycerol,20mMNa -EDTA,20mMEGTA,50mMNaF,5mMβ-glycerophosphate,
2
1mMphenanthroline,0.6%(w/v)PVP,and10mMascorbicacid). Thehomogenatewascentrifuged
at 2500ˆg for 20 min. After filtering through Miracloth, the supernatant was ultracentrifuged at
1500ˆgfor30min. Theresultantmicrosomalpelletwasresuspendedinresuspensionmedium(5mM
phosphatebuffer(pH7.8),330mMsucrose,2mMDTTand10mMNaF).Themicrosomalfraction
wasdistributedin27gphasesystemscontaining6.4%dextran,6.4%PEG(3350),5mMphosphate
buffer(pH7.8),2.5mMKCland300mMsucrose. Theplasmamembrane-enrichedfractionswere
retrievedfromtheupperphaseanddilutedinwashingbuffer(10mMTris,10mMboricacid,300mM
sucrose,9mMKCl,5mMNa -EDTA,5mMEGTA,and50mMNaF).Thesuspensionwascentrifuged
2
at3100ˆgfor45minat4˝Ctoobtainthefraction.
2.6. IsolationofCytosolicProteins
Cytosolicproteinswereextractedfrom1goftissueinlysisbuffercontaining26mMTris-HCl,2M
thiourea,0.3%(v/v)TritonX-100,20mMTrizmabase,7.5Murea,63mMCHAPS,andtheprotease
inhibitor cocktail complete mini (Roche, Mannheim, Germany). After shaking for 30 min at 4 ˝C,
7mMofDTTwasaddedtotheextract. Theproteinextractswerestirredfor20minandcentrifuged
twiceat20,000ˆgfor20minat4˝C.Thefinalsupernatantwasprecipitatedinonevolumeofacetone
containing20%TCA,9mMDTTat´20˝Covernight,thencentrifugedat20,000ˆgfor20minat4˝C.
Thepelletwasresuspendedintwovolumesofacetonecontaining9mMDTTat´20˝Cfor1h,then
centrifugedasabovetoobtainthecytosolicfraction[18].
2.7. EnzymeAssay
Catalaseandvanadate-inhibitedH+ATPaseactivitieswereassayedasdescribed[15]. Forcatalase
assay,thereactionmixturewaspreparedusing10µgeachofECMandcytosolicproteinsbyadding
50 µL of protein extract to 940 µL of 70 mM potassium phosphate buffer (pH 7.5). Reaction was
started by addition of 25 mM H O and a decrease in absorbance at 240 nm was monitored for
2 2
5min. BaselinecorrectionwasdonebysubtractingtheabsorbancetakenwithoutadditionofH O .
2 2
Theassaywasperformedintriplicatesandtheabsorbancevaluesobtainedwereplottedagainsttime.
Thevanadate-inhibitedH+ATPasewasassayedwith20µgofproteinforECMandplasmamembrane
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fractionin120µLofassaybuffer. Theassaywasperformedinthepresenceandabsenceof0.1mM
orthovanadate,freshlypreparedandboiledinbufferpriortoadditionof0.05%TritonX-100. Theassay
wasperformedinthepresenceofadetergenttoexposeallhiddensitesofthecellwallboundplasma
membrane, if any. The reaction was initiated by addition of ATP and was incubated at 37 ˝C for
30min. BlankslackingMgSO wereruninparallel[19]andthereleasedinorganicphosphatewas
4
determined[20]. Therelativeactivityoftheenzymewascalculatedbytakingthedifferencebetween
theabsorbanceat700nminabsenceandpresenceoforthovanadate.
2.8. Two-DimensionalPolyacrylamideGelElectrophoresis
TheECMproteinsweredilutedindilutionbuffer(100mMTris-Cl(pH8.5),20%(v/v)glycerol,
4%(w/v)SDS,20mMDTTand1mMPMSF)andboiledfor5min. Theproteinsamplewasallowedto
cooltoroomtemperature(25˝C),precipitatedwithninevolumesof100%chilledacetoneovernightat
´20˝C.Theprecipitateswererecoveredbycentrifugationat10,000ˆgat4˝C,for10min. Protein
pelletswerewashedtwicewith80%acetonetoremoveexcessSDS,air-driedanddissolvedin2-D
rehydrationbuffer. Rehydrationwascarriedoutfor16hwith300µgproteinresuspendedin200µL
rehydrationbufferand1000µgproteinresuspendedin450µLrehydrationbufferfor13cmand24cm
IPG strips, respectively. The rehydrated proteins were first separated by isoelectric focusing (IEF)
usinggelstripstoformanimmobilizedpHgradient(ImmobilineDryStrip,pH4–7,13cmandpH
4–7,24cm;GEHealthcare,ChalfontStGiles,UK)andthenbySDS-PAGEusing12.5%polyacrylamide
gels. IsoelectricfocusingwasperformedusingIPGphorsystem(AmershamBiosciences,Bucks,UK)
at 20 ˝C up to 30,000 Vhr for 13 cm and 80,000 Vhr for 24 cm IPG strips. The focused strips were
reducedwith1%(w/v)DTTin15mLofequilibrationbuffer(6Murea,50mMTris-HCl(pH8.8),30%
(v/v)glyceroland2%(w/v)SDS)for30min,followedbyalkylationwith2.5%(w/v)iodoacetamide
in the same buffer for 30 min. For 2-D SDS PAGE, the strips were placed on top of 12.5% (w/v)
acrylamidegelsalongwithlowrangeunstainedmarkers(Bio-Rad)atbasicendandPeppermintStick™
Phosphoproteinmarkers(Invitrogen,Carlsbad,CA,USA)atacidicend. Theelectrophoresedproteins
werestainedtovisualizephosphoproteinsandtotalECMproteinssequentially.
2.9. DetectionofECMPhosphoproteinsandProteins
Inordertodetectthephosphoproteins,2-Dgelswerefixedovernightinfixationsolution(50%
methanol,10%glacialaceticacid)andwashedwithdeionizedwaterthreetimeseachfor30minon
shaking. ThegelswerestainedwithPro-QDiamondphosphoproteingelstainsolution(Invitrogen,
Carlsbad, CA, USA) for 2 h on an orbital shaker and destained using destaining solution (20%
acetonitrile,sodiumacetate(pH4)(Sigma-Aldrich,St. Louis,MO,USA),threetimeseachfor30min)
followedbywashingwithdeionizedwaterthreetimeseachfor10min. Aftertakingtheimage,the
samegelswerethenstainedwithasilverstainpluskit(Bio-Rad)todetecttotalECMproteins.
2.10. ImageAcquisitionandinSilicoAnalysis
After 2-DE and gel staining, the Pro-Q Diamond stained gels were scanned using a Typhoon
scanner9210(GEHealthcare,ChalfontStGiles,UK)withanemissionwavelength580nmandsilver
stainedgelswerescannedbyaFluorSsystemequippedwitha12-bitcamera(Bio-Rad). Atleastthree
2-DEgelsrepresentingthreebiologicalreplicateswereincludedinthedataanalysis. Thescanned
gelimageswereprocessedandanalyzedwithPDQuestgelanalysissoftwareversion7.2.0(Bio-Rad).
Allimagesweresubsequentlyprocessedusingthesoftwaresettingsforspotdetection,background
subtraction,spotquantitation,andgel-to-gelmatchingofspotpatternsasdescribedpreviously[21].
Eachspotincludedinthestandardgelmetthecriteriaofbeingqualitativelyconsistentinsizeandshape
inthereplicategelsandbeingwithinthelinearrangeofdetection. Afewlandmarksinthegelseries
weremanuallydefinedtoimprovetheautomatedmatchingresults. Inaddition,forquantification
(based on spot density and area), the PDQuest software was used to assign quality scores to each
gelspot. Low-qualityspots(qualityscore<30)wereremovedbeforefurtheranalysis. Spotswith
aqualityscoregreaterthan30thatmettheabove-mentionedstringentcriteriawerethereafterreferred
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toashigh-qualityspots. Thehigh-qualityspotswereusedtocalculatethemedianvalueforagiven
spotandthisvaluewasusedasthespotquantityonthestandardgel. Thecorrelationcoefficientwas
maintainedataminimumof0.8betweengelimagesforahighlevelofreproducibility(FigureS1).
Next,forcomparison,theproteinspotswerenormalizedtothe“totaldensityingelimage”modeand
spotsweremanuallyannotated. ExperimentalmolecularmassandpIwerecalculatedfromdigitized
2-DEimagesusingstandardmolecularmassmarkerproteins.
2.11. PeptidePreparationforTandemMass-Spectrometry
Proteinspotsweremechanicallyexcisedfromsilver-stainedgelsandin-geldigestedwithtrypsin,
andpeptideswereextractedaccordingtostandardtechniques[22]. Inbrief, theexcisedgelplugs
werewashedtwicewithdeionizedwateranddestainedusing100mMsodiumthiosulfateand30mM
potassiumferricyanidesolution(1:1v/v)for15minwithshaking. Thegelpieceswerewashedtwice
in25mMammoniumbicarbonatein50%ACNwithgentleshakingfor10min. Thegelpieceswere
thendehydratedbysoakingin100%acid-freeACNfor5minandvacuumdriedfor20to30min.
Trypticdigestionwasperformedbyincubatingthegelpiecesin10µLoftrypsin(10ng/µL)overnight
at30˝C.Thedriedgelpiecesweredigestedwith0.2ng/µLcoldtrypsin(Sigma-Aldrich,St. Louis,
MO,USA)preparedin25mMammoniumbicarbonateinultrapurewater. Subsequently,thegelpieces
wereincubatedonicefor10mintoallowgelpiecestoswellandabsorbthetrypsinsolution,then
25µLof25mMammoniumbicarbonateinultrapurewaterwereaddedtocovergelpieces. Samples
wereincubatedat30˝Covernightwithshaking. Theextractedpeptideswerecollectedinafreshtube.
Tofurtherextractpeptides,10µLof1%(v/v)trifluoroaceticacidsolutionwereaddedtoeachgelpiece
andincubatedfor5min. Extractionsolutionwasremovedandaddedtodigestionmixture.
2.12. ProteinIdentificationUsingLC-MS/MS
ForLC-MS/MSanalysis,trypsin-digestedelutedpeptideswereloadedontoaC18Pep-Map100
column (3 mm, 100 Å, 75 mm ID_15 cm; LCPackings (Dionex, Thermo Fisher Scientific, Waltham,
MA,USA))at300nLmin´1,usingalineargradientofwater/acetonitrile/0.1%formicacid(v/v)and
analyzedbyelectrosprayionizationusingtheUltimate3000NanoHPLCsystem(Dionex,Thermo
Fisher Scientific) coupled to a 4000 QTRAP mass spectrometer (Applied Biosystems/MDS SCIEX,
FosterCity,CA,USA).Thepeptideswereelutedwithagradientof10%to40%acetonitrilewith0.1%
formicacid(v/v)over60min. TheMS/MSdatawereextractedusingAnalystsoftwareversion1.5.1
(AppliedBiosystems). Peptideanalysiswasperformedthroughdata-dependentacquisitionofmass
spectrometryscans(mass-to-chargeratiofrom400to1800),followedbyMS/MSscans. Peptideswere
identifiedbysearchingthepeaklistagainsttheViridiplantae(GreenPlants)(4,026,576sequences)with
NCBInrdatabase(8,994,603sequences; 307,8807,967residues)usingtheMascotversion2.1search
engine(www.matrixscience.com,MatrixScience,Boston,MA,USA).
Thedatabasesearchcriteriawereasfollows: taxonomy,allentries; peptidetolerance,˘1.2D;
MS/MS tolerance, ˘0.6 D; peptide charge, +1, +2, or +3; maximum allowed missed cleavage, 1;
fixedmodification,Cyscarbamidomethylation;variablemodification,Metoxidation;andinstrument
type, electrosprayionization(ESI)-iontrap. Thescorethresholdtoachievep<0.05wassetbythe
Mascotalgorithmandwasbasedonthesizeofthedatabaseusedinthesearch. Weconsideredonly
thoseproteinspotswhosemolecularweightsearchscorewasabovethesignificantthresholdlevel
determinedbyMascot. Forthenumberofobservedpeptidesperprotein,theuniquesequenceswere
counted and were imported to Excel spreadsheets (Table S1). For proteins identified by only one
peptidehavingascorehigherthan40thepeptidesequencewassystematicallycheckedmanually
(TableS2).
2.13. NetworkVisualization
Protein-protein interactions (PPI) were searched against geneMANNIA [23], BAR [24],
STRING[25],menthe[26],Interoporc[27],IntAct[28],DIP[29],APID[30],MINT[31],andBIND[32]
Proteomes2016,4,20 6of22
PPIdatabasesandtheinsilicogeneratedproteinandphosphoproteinnetworkwasvisualizedwith
Cytoscape[33]version3.0.2[34].
2.14. BioinformaticAnalysis
TheproteinfunctionswereassignedusingproteinfunctiondatabasePfamorInterProorthrough
literature survey. Since the functional annotation was based on Pfam and InterPro, the functional
redundancy was, thus, greatly minimized. The identified proteins were divided into different
functionalclassesaccordingtogeneontology(GO)andliterature. TheBLASTPsearchofidentified
proteinsequenceswasperformedthroughBlast2GO[35]againsttheUniprotproteindatabasewith
a minimum expectation value of 1 ˆ 10´3. Annotations were retrieved with default parameters:
pre-eValue-Hit-Filterat1ˆ10´6,cut-offwassetat55andGOweightat5. Tounderstandthefunction
ofunknownproteins,weperformeddomainanalysistopredicttheconserveddomain(s)usingthe
InterProScan[36]database. Thesubcellularlocalizationoftheputativephosphoproteinswaspredicted
usingTargetP[37],WolfPSORT[38]andYLoc+[39]programs.NetPhos2.0[40]analysiswasperformed
topredicttheputativesite(s)ofphosphorylation.
3. Results
3.1. EvaluationofPotatoECMIntegrityandPurityAssessment
Toconstructa2-DEmapofthepotatoECMproteoforms,weisolatedtheECMproteinfraction
by mechanical disruption and fractionation processes. The ECM-enriched pellet was washed
repeatedlyandextractedwithlow-osmolaritybuffertoremoveanycontaminantsfromotherorganelles.
ThecompletecellbreakagewasconfirmedbyanalysisofisolatedECMfractionusingastereozoom
microscope. The micrographs showed that the ECM fraction was free from plasma membrane or
otherultrastructuralcytoplasmicorganellesandthattherewerenointactcellswhichhadescaped
breakageduringhomogenization(Figure1A).ThepurityoftheECMwasevaluatedfurtherbyassaying
organelle-specificmarkerenzymeactivitiesthatcouldpossiblycontaminatethepreparation. Catalase
activitywasassessedforperoxisome,whilevanadate-inhibitedH+ATPaseactivitywasdetermined
fortheplasmamembranecontamination[19]. Thecrudecellextracts(ECMpelletbeforewashing)
showedhighcatalaseandATPaseactivities,whereastheECMfractiondidnotshowanysignificant
Proteomes 2016, 4, 20 7 of 26
activitiesofboththeenzymes(Figure1B,C).Theseresultsaltogethersuggestthatcytosolicandplasma
membranecontaminationsoftheECM,ifany,werebeyonddetectablelimits. Thus,inourstudy,the
contaminations of the ECM, if any, were beyond detectable limits. Thus, in our study, the purity of the
pEuCrMit ywoafs tchaereEfuCllMy cwonassidcaerreedfu flolyr fcuornthseidr earneadlyfsoers.f urtheranalyses.

Figure1. AnalysisofisolatedpotatoECMfractionanddeterminationofitspurity. (A)Micrograph
Figure 1. Analysis of isolated potato ECM fraction and determination of its purity. (A) Micrograph of a
toofluaidtionleu ibdluinee-stbaliuneed-s tsaeincteiodn seocf tipoontaotof pleoatfaletot lsehaoflweitngs htohwe inEgCMth espEeCciMficistyp eocfi fitchiety sotafinthineg.s t(aBi)n ing.
D(Be)teDrmetineramtioinn aotfi ocnataolfascea tsapleacsiefics paecctiivfiitcya icnt ipvoittyatoin EpCoMta taondE CcyMtosaonlidc fcryatcotisoonl.i cTfhrea cctyitoonso.liTch feraccytitoons olic
pfrraepctairoend fprormep aproetdatof rtiosmsue pfoort actaotaltaisses uaectivfoitry wcaatsa luasseed aasc taiv pitoysitiwvea scountsreodl. (aCs) Daetperomsiitniavteionc oonf trol.
v(Can)aDdeattee-rimnhinibaittieodn Hof+ vAaTnPaadsae taec-itnivhiitby itined thHe+ pAotTaPtoa sEeCaMct iavnitdy pinlasthmeap moteamtobrEaCneM fraancdtiopnl.a sTmhea pmlaesmmbar ane
mfreamctbiorann.eT fhraectpiolans omf apomtaetmo wbraasn uesferda catsi oan poosfitpivoet actoonwtroals. Dusaetad raespraespeonstsit eivxepecroimnternotls. pDeraftoarmreepdr einse nts
terxippleicraitme eanntds ipnedrifcoartemde ads i±n StDri.p licateandindicatedas˘SD.
3.2. Construction of 2-DE ECM Proteome and Phosphoproteome Map
ECM proteins and phosphoproteins were separated by 2-DE to establish a reference map. Initially,
the ECM proteome and phosphoproteome was resolved onto a 13 cm pH 3–10 that revealed more than
250 spots. However, proteins in the basic range exhibited poor resolution (Figure S2A). To make the
reference map well resolved, we used 13 cm narrow pH range (pH 4–7) that showed distribution of
most of the protein spots in the pI range of 4–7 and the molecular mass range of 14 to 100 kDa with
vertical streaking (Figure S2B,C). The reference map was made more comprehensive and vertical
streaking was reduced by using 24 cm IPG strips of pI range of 4–7 as the number of spots increased
with strip length and the resolution of spots was enhanced using 24 cm gels (Figure 2).
Proteomes2016,4,20 7of22
3.2. Constructionof2-DEECMProteomeandPhosphoproteomeMap
ECMproteinsandphosphoproteinswereseparatedby2-DEtoestablishareferencemap. Initially,
theECMproteomeandphosphoproteomewasresolvedontoa13cmpH3–10thatrevealedmore
than250spots. However,proteinsinthebasicrangeexhibitedpoorresolution(FigureS2A).Tomake
thereferencemapwellresolved,weused13cmnarrowpHrange(pH4–7)thatshoweddistribution
of most of the protein spots in the pI range of 4–7 and the molecular mass range of 14 to 100 kDa
withverticalstreaking(FigureS2B,C).Thereferencemapwasmademorecomprehensiveandvertical
streakingwasreducedbyusing24cmIPGstripsofpIrangeof4–7asthenumberofspotsincreased
withstriplengthandtheresolutionofspotswasenhancedusing24cmgels(Figure2).
Proteomes 2016, 4, 20 8 of 26

Figure 2. ECM proteome and phosphoproteome profiles. (A) ECM proteome profile of silver stained 2-
Figure2.ECMproteomeandphosphoproteomeprofiles.(A)ECMproteomeprofileofsilverstained
DE gels. (B) ECM phosphoproteome profile of Pro-Q stained 2-DE gels. The experiments were carried
2-DEgels.(B)ECMphosphoproteomeprofileofPro-Qstained2-DEgels.Theexperimentswerecarried
out in triplicates and further computationally combined into a representative standard gel. Each spot is
outiinndtirciaptleidc abtye sreadn adrrfouwrt hanedr caoccmompuptaantiieodn waliltyh csopmot bniunmedbeirn btoraackreetpedre wseitnht aMtiwv eanstda pnId. ardgel.Eachspotis
indicatedbyredarrowandaccompaniedwithspotnumberbracketedwithMwandpI.
More than 329 protein spots and 88 phosphoprotein spots were detected on 13 cm gels pI 4–7, out
of which 300 protein spots and 79 phosphoprotein spots survived the filtering process. While 24 cm pH
4–7 strips showed 733 protein spots and 259 phosphoprotein spots, with 704 high-quality protein spots
Proteomes2016,4,20 8of22
Morethan329proteinspotsand88phosphoproteinspotsweredetectedon13cmgelspI4–7,out
ofwhich300proteinspotsand79phosphoproteinspotssurvivedthefilteringprocess. While24cm
pH4–7stripsshowed733proteinspotsand259phosphoproteinspots,with704high-qualityprotein
spotsand241phosphoproteinspotsmatchedtoourfilteringcriteria. Thespotswerenumberedas
StEP-1toStEP-733andStEPP-1toStEPP-259(theletteres‘St’identifytheorganism(Solanumtuberosum)
and‘E’signifiesthesubcellularorganelle(ECM)fromwhichtheproteome‘P’andphosphoproteome
‘PP’maphasbeenmade,whereasthenumeralsindicatethespotnumbers(Figure2A,B).
3.3. DistributionPatternandPhysicochemicalCharacteristicsofECMProteinsandPhosphoproteins
Atotalof38high-qualityproteinspotsand35phosphoproteinspotswereexcisedandidentified
byiontrapLC-MS/MSFigureS3,Tables1and2). Venndiagramshowedthatfouridentifiedproteins
andphosphoproteinswerecommonbetweenthetwodatasets(FigureS4). Onthebasisoftheaverage
quantitativeexpressiondata,ECMproteinsandphosphoproteinswerecategorizedintothreemajor
classes: highabundance(35.0%;>1%–2%ofthetotalproteinload,and67.74%;>1%–2%ofthetotal
phosphoproteinload,andmediumabundance(20.6%;0.5%–1%ofthetotalproteinload,and17.74%;
0.5%–1%ofthetotalphosphoproteinload). Lowabundantproteinsaccountfor44.6%(0.03%–0.5%of
thetotalproteinload)and14.5%(0.03%–0.5%forthephosphoproteinload).
Proteomes 2016, 4, 20 15 of 26
Toexamineputativecharacteristicfeatures,theidentifiedproteinsandphosphoproteinswere
To examine putative characteristic features, the identified proteins and phosphoproteins were
analyzedwithrespecttoexperimentalmolecularmassandpIdistribution. 2-DEmapofECMwas
analyzed with respect to experimental molecular mass and pI distribution. 2-DE map of ECM was
dominated by proteins having molecular weight of 0–50 kDa (60%) followed by 50–75 kDa and
dominated by proteins having molecular weight of 0–50 kDa (60%) followed by 50–75 kDa and 75–100
75–100kDa(Figure3A).ThemajorityofproteinsexhibitedadistributionpatternrangedfrompI5.5–6.5
kDa (Figure 3A). The majority of proteins exhibited a distribution pattern ranged from pI 5.5–6.5 (65%)
(65%)followedbypI4.5–5.5(Figure3B).Furthermore,physicochemicalcharacteristicsrevealedthat
followed by pI 4.5–5.5 (Figure 3B). Furthermore, physicochemical characteristics revealed that
phopshpohsopphrooptreoitnesinssh sohwoweeddp prreeddoommiinnaannccee inin mmoloecleuclaurl warewigheti,g rhatn,grianngg frinomg f2r5o–m50 2k5D–a5 a0nkdD paI 5a.n5–d6.p5I. 5.5–6.5.

Figure 3. Physicochemical characteristics of proteins and phosphoproteins. Distribution of proteins and
Figure3.Physicochemicalcharacteristicsofproteinsandphosphoproteins.Distributionofproteins
phosphoproteins based on (A) molecular weight (Mw) and (B) Isoelectric point (pI).
andphosphoproteinsbasedon(A)molecularweight(Mw)and(B)Isoelectricpoint(pI).
3.4. Functional Categorization of ECM Proteins
To comprehend the function of the ECM proteins, the identified proteins were sorted into seven
categories, as shown in Figure 4A. However, the classification of proteins is only tentative, since the
biological function of many proteins identified has not yet been established experimentally. About 26%
of the identified proteins were grouped under the unknown category, since no information as to their
potential function in the organelle was available. Domain analysis of unknown proteins revealed that
HAD-like domain, NAD(P)-binding domain, inorganic pyrophosphatase were identified in ECM
proteome (Table S3). Proteins involved in metabolism (32%) were most abundant in our study. StEP 529
and 675 were identified to be alpha-1,4-glucanprotein synthase (UDP-forming). It is an ECM remodeler
that is involved in architecture reformation and restructuring. Two isoforms of ErbB-3 binding protein
(EBP1), StEP 1005 and 1213, were also present in the potato ECM proteome. EBP1 is known to function
as a signal component in wall hydration [41]. 5-methyltetrahydropteroyltriglutamate homocysteine
Proteomes2016,4,20 9of22
Table1.ListofidentifiedECMproteinsofpotatobyMS/MSanalysis.
Functional
SpotIDa. ProteinName Species ProteinIDb Score NPc %coverage Thr.Mw/pI Exp.Mw/pI
Category
StEP-840 Enolase Lycopersiconesculentum 119354 88 3 10 47.768/5.68 60.334/5.49
5-ethyltetrahyd
StEP-1003 ropteroyltriglutamate- Solanumtuberosum XP_002328083.1 198 5 6 84.54/6.26 53.065/5.82
homocysteine
Ferredoxin-NADP(H) Oryzasativa(japonica
StEP-1166 41052915 57 2 5 40.638/7.98 40.844/6.22
oxidoreductase cultivar-group)
StEP-1418 Glutamatedehydrogenase Lycopersiconesculentum 12229803 441 9 26 44.78/6.68 53.599/6.94
Alpha-1,4-glucan-protein
Metabolism StEP-675 Solanumtuberosum 34582499 216 8 22 41.57/5.71 51.107/5.29
synthase
RGP3(Reversiblyglycosylated
StEP-529 Arabidopsisthaliana 30680679 69 2 4 41.25/5.39 50.666/4.94
polypeptide3)
StEP-376 Transaldolase Unknownspecies 85112172 78 2 6 35.20/5.38 45.309/4.62
StEP-1143 Proteinthylakoidformation1 Solanumtuberosum 75140959 140 5 20 33.34/8.69 28.646/5.89
StEP-1213 ErbB-3bindingprotein1 Solanumtuberosum 116292768 320 7 21 42.79/6.25 58.028/6.37
StEP-1005 ErbB-3bindingprotein1 Solanumtuberosum 116292768 147 5 13 42.79/6.25 58.891/5.87
DHputative
StEP-893 beta-hydroxyacyl-ACP Capsicumannuum 193290688 138 4 14 23.966/9.35 22.904/5.64
dehydratase
StEP-1333 Germin-likeprotein1 Nicotianatabacum 1169944 58 1 4 21.98/6.27 26.698/6.75
StEP-1122 24Kgerminlikeprotein Nicotianatabacum 31711507 67 2 15 21.954/5.82 26.698/6.75
Development
ChainA,TheCrystalStructure
StEP-656 Solanumtuberosum 31615943 72 2 4 40.93/5.17 39.366/5.25
OfSemetPatatin
ChloroplastDrought-induced
Stress StEP-962 Solanumtuberosum 2582822 221 7 20 33.436/8.07 37.4815.64
StressProteinof32kDa
Response
ChloroplastDrought-induced
StEP-965 Solanumtuberosum 2582822 179 7 20 33.436/8,07 37.525/5.83
StressProteinof32kDa
StEP-567 Chaperonin-60betasubunit Solanumtuberosum 1762130 275 7 17 62.98/5.72 76.934/4.85
Protein StEP-405 Chaperoninhsp60 Arabidopsisthaliana 16221 279 7 12 61.31/5.66 70.819/5.12
Folding StEP-402 Heatshockprotein70 Nicotianatabacum 30025966 457 8 13 70.83/5.17 76.624/5.06
StEP-1024 Leucineaminopeptidase2 Lycopersiconesculentum 2492530 191 5 11 59.512/8.18 69.045/5.67
StEP-1417 GTP-bindingprotein Capsicumannuum 7643796 433 12 38 44.37/6.45 57.878/6.88
Signaling StEP-1001 Glucose-regulatedprotein78 Lycopersiconesculentum 170386 177 5 12 41.15/8.51 55.669/5.78
StEP-810 Endoplasminhomolog Hordeumvulgare 544242 52 1 1 92.859/4.86 41.936/5.47
Proteomes2016,4,20 10of22
Table1.Cont.
Functional
SpotIDa. ProteinName Species ProteinIDb Score NPc %coverage Thr.Mw/pI Exp.Mw/pI
Category
Dehydroascorbatereductase-like
StEP-1137 Solanumtuberosum 76160951 62 2 9 23.425/6.09 28.647/5.99
protein
Redox StEP-922 Dehydroascorbatereductase Lycopersiconesculentum 66475036 61 2 9 23.523/6.32 28.508/5.79
Homeostasis StEP-615 Thioredoxinperoxidase1 Lycopersiconesculentum 30841938 57 2 11 17.426/5.18 20.169/5.68
StEP-765 Peroxiredoxin Ricinuscommunis 223543823 55 1 5 21.82/8.74 22.953/5.42
StEP-530 Lactoylglutathionelyase Ricinuscommunis 223542315 249 4 16 31.52/7.63 42.983/4.98
StEP-848 Unknownprotein18 Pseudotsugamenziesii 205830697 63 1 91 1.393/5.80 70.364/5.37
StEP-853 Unknownprotein18 Pseudotsugamenziesii 205830697 50 2 100 1.393/5.80 79.178/5.508
StEP-1004 Unknownprotein18 Pseudotsugamenziesii 205830697 83 2 100 1.39/5.80 58.661/5.83
StEP-352 Unknownprotein18 Pseudotsugamenziesii 205830697 99 2 100 1.39/5.80 40.823/4.82
StEP-879 Unknownprotein19 Pseudotsugamenziesii 205830698 54 1 91 1.393/5.80 13.226/5.69
Unknown
StEP-1180 Unknown Solanumtuberosum 77745483 281 7 20 37.64/7.70 45.963/6.01
StEP-354 Unknown Populustrichocarpa 118486367 71 3 8 44.55/8.48 41.326/4.72
StEP-963 Hypotheticalprotein Pseudotsugamenziesii 83649448 55 2 2 36.547/6.47 37.799/5.78
StEP-346 Predictedprotein Populustrichocarpa 224103823 113 2 7 33.55/6.85 38.700/5.02
Oryzasativa(japonica
StEP-638 Os03g0822200 115456265 57 1 6 27.89/6.34 33.319/5.17
cultivar-group)
aSpotnumberasmarkedontheECMproteome2-DgelimageFigure2A.ThespotnumbersweredesignatedasStEP,whereStindicatestheorganismSolanumtuberosum,EPdenotes
theExtracellularMatrixProteome.bProteinidentificationnumberasingenebank.cNumberofpeptides.