Experimental
setup
for
the
identi
fication
of
mitochondrial
protease
substrates
by
shotgun
and
top-down
proteomics
Alice
Di
Pierro
a,
Heather
Bondi
a,b,
Chiara
Monti
a,b,
Luisa
Pieroni
c,d,
Enrico
Cilio
c,d,
Andrea
Urbani
c,d,
Tiziana
Alberio
a,b,*
,
Mauro
Fasano
a,b,*
,
Maurizio
Ronci
eaDepartmentofScienceandHighTechnology,UniversityofInsubria,I-21052BustoArsizio,Italy b
CenterofNeuroscience,UniversityofInsubria,I-21052BustoArsizio,Italy
c
SantaLuciaIRCCSFoundation,I-00143Rome,Italy
d
DepartmentofExperimentalMedicineandSurgery,UniversityofRome“TorVergata”,I-00133Rome,Italy
e
Dept.ofMedical,OralandBiotechnologicalSciences,University“G.D'Annunzio”ofChieti-Pescara,I-66013Chieti,Italy
ARTICLE INFO Articlehistory:
Received18November2015
Receivedinrevisedform25January2016 Accepted16February2016
Availableonline22February2016 Keywords: Mitochondria Protease Shotgun Top-down Electroelution ABSTRACT
Mitochondriapossessaproteolyticsystemthatcontributestotheregulationofmitochondrialdynamics, mitochondrialbiogenesisandmitophagy.Weaimedattheidentificationbybottom-upproteomicsof altered protein processingdue totheactivationof mitochondrialproteases in acellular modelof impaireddopaminehomeostasis.Moreover,weoptimizedtheconditionsfortop-downproteomicsto identifythecleavagesitesequences.
ã2016TheAuthors.PublishedbyElsevierB.V.onbehalfofEuropeanProteomicsAssociation(EuPA).This isanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/
4.0/).
Proteomicsandgenomicsinvestigationsfortheidentification andcharacterizationofproteasesandtheirsubstratesareglobally referredtoasdegradomics.Proteasesarenotonlyinvolvedinthe degradationofunfoldedordamagedproteins,butservetoprovide asystembywhichproteinfunctionisregulated.Indeed,proteases catalyzeanirreversiblehydrolysisreactionregulatingactivityand localisationofmanyproteins[1].Mitochondriapossessahighly conserved, intraorganelle proteolytic system that conducts the surveillanceof proteinquality controlwithin mitochondria[2]. These proteases degrade damaged or unfolded proteins to peptides, which are subsequently either transferred out of the organelle orfurtherdegradedbymitochondrial oligopeptidases
[3]. Inadditiontotheirconventionalrole inthedegradationof misfoldedordamaged mitochondrial proteins,many mitochon-drial proteases take part in the regulation of proteins that orchestrate mitochondrial dynamics, mitochondrial biogenesis and mitophagy [4]. An alteration of mitochondrial proteases activityisdetrimentaltocellhealthandhasbeenlinkedtoaging
and to various diseases, including neurological disorders like spinocerebellarataxiaandParkinson'sdisease(PD)[5,6].Indeed, mutationsinthemitochondrialserineproteaseHTRA2havebeen associatedwithPDinsporadicpatients[7].Wepreviouslyreported a selective degradation of voltage-dependent anion-selective channel proteins (VDACs) induced by dopamine toxicity, in a cellularmodelofPD[8].However,bythecombinationofgel-based andgel-freetechniques,weobservedanaccumulationofVDACs proteolyticfragmentsinsidemitochondria,togetherwith proteo-lytic fragments belonging to other mitochondrial proteins [9]. These findings suggested that the impairment of dopamine homeostasis, one of the putative causes of sporadic PD, could induceanalterationofmitochondrialproteasefunctions,deeply compromising themitochondrial quality control systems.From this point of view, a more comprehensive characterization of mitochondrial proteases could provide new insights into PD pathogenesis.
This work was developed in the contextof the biology and diseaseactionofthemitochondrialhumanproteomeproject (mt-HPP) [10]. To understand protease functions it is of primary importancetodeterminetheirsubstratesandtheircleavagesite(s). Inordertoidentifywhichsubstratesweredifferentiallyprocessed bymitochondrialproteasesinacellularmodelfortheimpairment of dopamine homeostasis [11], we combined an in-gel
* Corresponding authors at: Department of Science and High Technology, UniversityofInsubria,I-21052BustoArsizio,Italy.Fax:+390332395599.
E-mailaddresses:tiziana.alberio@uninsubria.it(T.Alberio),
mauro.fasano@uninsubria.it(M.Fasano).
http://dx.doi.org/10.1016/j.euprot.2016.02.002
2212-9685/ã2016TheAuthors.PublishedbyElsevierB.V.onbehalfofEuropeanProteomicsAssociation(EuPA).ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).
EuPAOpenProteomics11(2016)1–3
ContentslistsavailableatScienceDirect
EuPA
Open
Proteomics
fractionation of mitochondrial proteins, followed by shotgun proteomicsofselectedmolecularweightranges.
SH-SY5Yhumanneuroblastomacellsweretreatedornotwith 250
m
Mdopamine,inthepresenceof700U/mLofcatalasefor24h, in three independent replicates per condition. Mitochondrial enriched fractions were obtained by differential centrifugation accordingtostandardized guidelines developed bythe mt-HPP team[9].SampleswerelysedinRIPAbuffer(50mMTris-HClpH 7.6,150mMNaCl,1%sodiumdeoxycholate,1%NP-40and0.1%SDS) and30m
gofmitochondrialproteinswereseparatedbySDS-PAGE electrophoresisonacrylamide/bisacrylamide16%gel.Threeslices indifferentmolecularweightranges(i.e.,40–25,25–15and15– 10kDa) were manually excised (Fig. 1A), washed twice with 100mMNH4HCO3 in50% v/vacetonitrile (ACN)for 10min and subjectedtotrypsindigestion.Cysteineswerereducedwith10mM DTTin100mMNH4HCO3for45minat56Candalkylatedwith 55mM iodoacetamide (IAA) in 100mM NH4HCO3 at room temperaturefor30mininthedark.Gelsliceswerewashedtwice asdescribedaboveanddriedinavacuumcentrifuge.Thedrygel pieceswere incubated with digestion buffer(12ng/m
L porcine trypsin,100mMNH4HCO3,10mMCaCl2and5%ACN)overnightat 37C.Afterabriefcentrifugation,supernatantscontaininghydro-phylic peptides were saved in a new microcentrifuge tube. Hydrophobicpeptideswereextractedbyadding1%trifluoroacetic acid(TFA)andincubatingatroomtemperaturefor10minutesin agitation.Supernatantswerecollectedandpooledwiththefirst ones.Eventually,60%v/vACNand0.1%v/vTFAwereaddedtothe gelpiecesandafter10minutesvortexingthesupernatantswere collected,pooledtogetherandvacuumdried.Thedigestedsamples wereanalyzed by nano LC-MS/MS using a Proxeon EASY-nLCII (ThermoFisherScientific,Milan,Italy)coupledtothemaXisHD UHR-TOFmassspectrometer(BrukerDaltonics).Five
m
Lofsample wereinjectedandconcentratedonatrappingC18-A1EASYColumn (2cm,100m
mI.D.,5m
mp.s.,ThermoFisherScientific).TrappedpeptidesweresubsequentlyseparatedonaC18-AcclaimPepMap (25cm,75
m
mI.D.,5m
mp.s.,ThermoFisherScientific)at0.3m
L/ minwithagradientfrom2to45%Bin20min(eluents:A,0.1%FAin H2O; B, 0.1% FAin CH3CN).The raw datawereprocessed with PEAKS 7.5 and searched against UniProt SwissProt database (release 2015_03 including common MS contaminants 20,441 entries). The tolerances for the mass errorwere set to 15ppmand0.05Daforprecursorsandfragmentsrespectively,two missedcleavageallowed,carboamidomethylationofCysasfixed modificationandoxidationofMet,deamidationofAsnandGlnand acetylationofLyswereselectedasvariablemodifications. 10logP valueforpeptideswasadjustedtoprovideanFDRlowerthan0.1% atpeptideandproteinlevel.Weemployedabottom-upapproachtodetecttheproteinsthat were present at a lower molecular weight compared to their theoretical one, indicating a possible proteolytic processing. Amongallidentifiedproteolyticfragments,onlythemitochondrial 60kDaheatshockprotein(HSPD1)andthemitochondrialporin VDAC1showedadifferentbehaviourbetweendopaminetreated cells and controls. Although HSPD1 resulted processed by proteasesinbothconditions,withproteolyticfragmentspresent in all gel-fractions, dopamine induced an accumulation of HSPD1 peptides in the25–15kDa region(Fig.1B). In linewith ourpreviousreports[8,9],dopamineinduceda decreaseof full lengthVDAC1(30kDa)alongwithanaccumulationofVDAC1small peptides(inthe25–15kDafraction)insidemitochondria(Fig.1C). Since all peptides identified by shotgun proteomics were trypsindigested,wewerenotabletoreconstructtheaminoacidic sequenceofprocessedsubstrates,sotodetecttheconsensussiteof proteases.Toovercometheselimitationsandidentifyproteolytic fragmentsgenerated inthemitochondriaafterdopamine treat-ment,wemovedtowardsatop-downapproach,whereproteinsare separated by electrophoretic or chromatographic techniques without any prior digestion of the sample. However, the
Fig.1.Bottom-upanalysisofmitochondrialproteolyticfragments.(A)Schematicrepresentationofgelslicinginthreefractionswithdifferentmolecularweightrange(i.e., 40–25,25–15and15–10kDa).(B)HSPD1proteolyticpeptidelevelsinthe40–25kDafraction(bottom)and25–15kDa(top).(C)VDAC1proteolyticpeptidelevelsinthe40– 25kDafraction(bottom)and25–15kDa(top).CTR:cellsundercontrolconditions,DA:cellstreatedwith250mMdopamine.MeanandSEMrefertothreeindependent replicates.Forfurtherdetailsseetext.
measurement of intact proteins presents many technical chal-lenges, especially for complex multiprotein samples, thus we focusedourefforts onestablishing a reliableprocedure for the preparation of protein samples for top-down proteomics. We decidedtoseparateproteinsthroughSDS–PAGE,cutthinslicesat theleveloflowerMWandelectroeluteeachprotein(orfragments) out of thegel.To this purpose, a homemadeelectroeluter was designedandassembledusingapolymethylmethacrilatesupport, milledtoobtaintheelutioncell(120.2cm).Thecellwasthen drilledtoallowtheinsertionoftwoplatinumelectrodes,plugged toapowersupply.Electroelutionconditionsettingwasoptimized usingprestainedproteinMWmarkers(PageRulerPlusPrestained ProteinLadder,10to250kDa,Thermo-Scientific),separatedinan acrylamide/bisacrylamide16%gel.Proteinbandsweremanually excised,placedintheelutercellandcoveredwith200
m
Lelution buffer(500mMNH4HCO3).Byapplyingaconstantcurrent(10mA), weobservedthat20minwereneededtoelutethe25kDa protein-marker,25minforthe35kDaproteinand30minforthe70kDa protein.Thebufferwascollectedassoonasthegelbandsbecame colourlessindicatingthecompleteelutionoftheproteins.Inorder toremoveSDSresiduesthatcouldinterferewithsubsequentMS analysis,sampleswereaddedwith20%v/vethanolandloadedinto anultrafiltrationcell(Vivaspin500MWCO3000DaPES,Sartorius) and centrifugedat 15000gto a finalvolume of 50m
L. Then, retainedsampleswerecollectedinnewtubesandvacuumdried. Toverifytheefficiencyoftheproteinelution,driedsampleswere re-suspended in Laemmlibuffer and loadedon anacrylamide/ bisacrylamide16%gel.Afterblue-silverstaining[12],weobserved singleproteinbandsatthesamemolecularweightoftheeluted proteins, confirming that proteins were correctly eluted and recovered(Fig.2).Futureworkwillbedevotedtothefragmentationofisolated proteins to obtain sequence information. The availability of terminalsequencesofaproteolyticfragmentbytop-downanalysis couldbeusedtoidentifytheproteasepotentiallyresponsiblefor itsformationusingtheMeropsdatabase(http://merops.sanger.ac. uk/).Inthis database,fora specificproteasearereportedallits substrates and vice versa. The information in the database is retrievedfromtheliterature,thereforesomereporteddatamight nothavephysiologicalrelevance.
Asawhole,thepresentcommunicationdescribesastrategyfor theidentificationforcleavedproteasesubstratesbycombininga
shotgun analysisof pre-fractionatedlow-molecular-weight pro-tein components with electroelution of full-lengthproteins for top-down characterization. The proposed procedure should constitute a basis for both identification and quantification of proteins that migrate at a molecular weight lower than that expected, and theidentification oftheirsequence termini,thus permittingtheassessmentof putativeproteasesresponsiblefor theircleavage.
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Fig.2. Validationoftheelectroelutionprocedure.PrestainedproteinMWmarkers(70and55kDa)wereexcisedfromanacrylamide/bisacrylamide16%gel.After elecroelution,sampleswerecleanedfromSDSresiduesbyultrafiltration(MWCO3kDa)with20%v/vethanol.Sampleswerevacuumdried,resuspendedwithLaemmlibuffer andseparatedbySDS-PAGE.Firstlane:55kDamarker;secondlane:70kDamarker.
