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HISTOLO
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Pre-printauthor´sversion
ISSN:0213-3911e-ISSN:1699-5848
SubmityourarticletothisJournal(http://www.hh.um.es/Instructions.htm)
Fiber type diversity in skeletal muscle explored by mass spectrometry-based single fiber proteomics
Authors:Stefano Schiaffino, Carlo Reggiani and Marta Murgia DOI:10.14670/HH-18-170Articletype:REVIEWAccepted:2019-10-15Epubaheadofprint:2019-10-15
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Fibertypediversityinskeletalmuscleexploredbymassspectrometry-basedsinglefiberproteomicsStefanoSchiaffino1,CarloReggiani2,3andMartaMurgia2,41VenetianInstituteofMolecularMedicine(VIMM),Padova,Italy2DepartmentofBiomedicalSciences,UniversityofPadova,Padova,3InstituteforKinesiologyResearch,ScienceandResearchCenterofKoper,6000Koper,Sloveniaand4Max-Planck-InstituteofBiochemistry,Martinsried82152,GermanyAddressforCorrespondence:StefanoSchiaffino,VenetianInstituteofMolecularMedicine(VIMM),ViaOrus2,35129Padova,Italy.E-mail:[email protected]:SinglemusclefiberproteomicsKeywords:skeletalmuscle,musclefibertypes,singlemusclefiberproteomics,massspectrometry
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SummaryMammalianskeletalmusclesarecomposedofavarietyofmusclefiberswithspecialized
functionalproperties.Slowfibersaresuitedforlonglastingandlowintensitycontractileactivity,
whilevarioussubtypesoffastfibersareoptimizedtoproducehighforceandpowerevenwitha
significantfatigue.Thefunctionalspecializationofmusclefibersisbasedonselectivegene
expressionregulation,whichprovideseachfiberwithaspecificproteincomplement.
Therecentrefinementofsmall-scalesamplepreparation,combinedwiththedevelopmentof
massspectrometerscharacterizedbyhighsensitivity,sequencingspeedandmassaccuracy,has
allowedthecharacterizationoftheproteomeofsinglemusclefiberswithanunprecedented
resolution.Inthelastfewyears,thefirststudiesontheglobalproteomicsofindividualfibersof
differenttypeshavebeenpublished.Inthisshortreviewwediscussthemethodological
advancementswhichhaveopenedthewaytosinglefiberproteomicsandthediscoverypowerof
thisapproach.Weprovideexamplesofhowspecificfeaturesofsinglefiberscanbeoverlooked
whenwholemuscleormulti-fibersamplesareanalyzedandcanonlybedetectedwhenasingle
fiberproteomeisanalyzed.Thus,novelsubtype-specificmetabolicfeatures,mostprominently
mitochondrialspecializationoffibertypeshavebeenrevealedbysinglefiberproteomics.Inthe
sameway,specificadaptiveresponsesofsinglefiberstoagingorlossofneuralinputhavebeen
detectedwhensinglefiberswereindividuallyanalyzed.Weconcludethatthefibertype-resolved
proteomesrepresentapowerfultoolwhichcanbeappliedtoavarietyofphysiologicaland
pathologicalconditions.
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Introduction
Adistinctivefeatureofskeletalmuscleisthediversityandspecializationoffibertypes,which
allowmusclestofulfillavarietyoffunctionaltasksbyselectiverecruitmentofthemostsuitable
fibers:fatigueresistantslowfibersforlonglastingactivity,powerfulandfatiguablefastfibersfor
shortburstsofhighintensitywork.Biochemicalstudiesofselectedmuscleswithrelatively
homogeneousfibertypecomposition,whicharefoundinsomespecies,haveinitiallyprovided
somebasicinformationaboutthemolecularfeaturesofmuscletype-specificity.Forexample,the
seminalstudiesbyBarany(1967)basedonacomparativestudyoffastandslowmusclesfrom
differentspecies,revealedforthefirsttimetherelationshipbetweenmaximummyosinATPase
activityandthespeedofcontraction(seealsoMossandSolaro,2019).However,themixedfiber
typecompositionofmostskeletalmuscles,particularlyintwospecies,mouseandhuman,most
relevantforbiomedicalresearch,complicatestheinterpretationofbiochemicalstudiesatthe
wholetissuelevel.Dataderivedfromwholemusclestudiesreflectinfactboththeproportionof
thedifferentfibertypesandthevalueoftheparametersanalyzedineachfibertype.Inaddition,
wholemuscleanalysesalsoincludenon-musclecells,whichareaminorcomponentintermsof
globalproteincompositionbutamajorcomponentintermsofproteinspresentinthenucleus,as
myonucleirepresentlessthan50%oftotalnucleipresentinmuscletissue(Schmalbruchand
Hellhammer,1967).
Toaddressthisissueandidentifytheproteinsexpressedinspecificfibertypes,twoapproaches
weresubsequentlyused.Thefirstapproachisbasedonenzymehistochemistryand
immunohistochemistryanalysesofmusclesections.Thesecondapproachinvolvesthedissection
ofsinglefibersfromskeletalmusclesandtheanalysisoftheproteincompositionbySDS-PAGEand
Westernblotting.Acrucialstepinthestudyofmusclefibertypeswasthedemonstrationthat
skeletalmusclescontaindifferentmyosinheavychains(MYH)isoforms,whichcanbeidentifiedby
specificantibodiesandseparatedbyappropriateSDS-PAGEprotocols.MYHsthusbecamea
standardmarkertoidentifyfibertypesinmusclesectionsandinisolatedfibers.Thesestudiesled
tothecurrentviewthat,basedonMYHcomposition,mousemusclescontainfourmajorfiber
types,slowtype1andfast2A,2Xand2B,whilehumanmusclescontainthreemajorfibertypes,
slowtype1andfast2Aand2X(SchiaffinoandReggiani,2011).Additionalfibertypes,definedby
uniqueMYHisoforms,areconfinedtospecializedmuscles,suchascranialmuscles:forexample,
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slow-tonicfibers,whichrespondtostimulationwithacontractureratherthanatwitch,are
presentinextraocularmusclesbutnotinmosttrunkandlimbmuscles(Rossietal.,2010).
Two-dimensionalgelelectrophoresiscombinedwithmassspectrometry(MS)allowedtoidentifya
significantnumberofproteins,uptoafewhundred,inskeletalmusclesamples(seeMurphyetal.,
2016;Capitanioetal.,2017).However,manymuscleproteins,e.g.MYHs,cannotbeanalyzed
becausetheydonotenterthegelinthefirstdimension(isoelectricfocusing,IF).Inaddition,two-
dimensionalgelelectrophoresis,whichrequiresabout100µgofprotein,cannotbeappliedtothe
studyofsinglemusclefibers,whichcontainaround1µgofprotein.Thislimitationalsoappliesto
otherapproachesinvolvingtheisolationofspecificmusclefiberstructures,suchascontractile
proteinsormitochondria,andtheanalysisoftheseproteinsbyMS.Sarcomericprotein-enriched
extractsofskeletalmuscles,derivedfrom5mgofmuscletissue,havebeenanalyzedbytop-down
targetedproteomicstodeterminealternativesplicingandpost-translationalmodificationsof
contractileproteins(Jinetal.,2019).Furthermore,gel-basedtechniquesarelaboriousandrequire
longhands-ontimepersample,thuspreventingmultiplexingoftheproceduretohighsample
numbers.
Theproteomicanalysisofisolatedsinglemusclefiberswasmadepossiblebyrecentadvancesin
proteomicstechniques,inparticularthedownscalingofthesamplesizethroughthedevelopment
ofshotgunMSapproaches(seebelow),whichallowtheidentificationofthousandsofproteinsin
asinglemusclefiber.Inthisshortreviewwediscussthemethodologicalbasisofthisapproachand
themainresultsobtainedinstudiesonmouseandhumanskeletalmuscles.
Methodologicalaspects
Amajorchallengeencounteredintryingtoapplymodernproteomicstechniquestosinglemuscle
fibersistheverylowamountofproteinscontainedinasinglemusclefiberor,inthecaseof
humanmuscles,inasegmentoffiberascanbedissectedfromabiopsysample.Theestimated
amountofproteininasinglemusclefiberisolatedfromtypicalmousemuscles,suchastheslow
soleusorthefastextensordigitorumlongus(EDL)isabout0.5μg,assumingacrosssectionalarea
of1000μ2,alengthof5mmandacylindricalshape.Astohumansamples,duetothickercross-
sectionalarea,similarcalculationsleadto0.5μgpermmlengthandthusapproximately1.5μg
withanaveragelengthof3mminabiopsytakenwithaBergstromneedle.Theseminutesample
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amountsarenotsuitableforIF+SDS-PAGE-basedapproaches.Theproblemhasbeenaddressed
byoptimizinganin-solutiondigestionworkflow,wherebyallpreparationsteps,fromsamplelysis
totrypsindigestion,arecarriedoutinonebufferandconfinedtoasinglevessel.Thelatteris
subsequentlyalsousedforpeptidepurification,thusminimizingsampleloss(Kulaketal.,2014).In
ourstudiesweappliedthisapproachtosinglefibers,whichweremanuallyisolatedand
immediatelysnap-frozen(Murgiaetal.,2015and2017).Othergroupshaveusedcollagenase
digestionformusclefiberisolation(Langetal.,2018).
Asimplifiedworkflowofthesingle-fiberproteomicsusedinourstudiesispresentedinFig.1.An
indicationoftherelativeconcentrationofthevariousproteinsinthemusclefiberscanbederived
fromthecorrespondingIBAQ(Intensity-BasedAbsoluteQuantification)values,anoutputofthe
MaxQuantanalysissoftware(CoxandMann,2008;Tyanovaetal.,2016).IBAQvaluesaretheMS
signalintensitiesdividedbythenumberoftheoreticalpeptides,thusareroughlyproportionalto
themolarquantitiesoftheproteins.InourstudiesIBAQvaluesofindividualproteinswere
normalizedtotheIBAQvalueofα-skeletalactintoaccountforthevariablecross-sectionalarea
andlengthofthefibersanalyzed.
Afurthermethodologicalchallengeinsingle-fiberproteomicsisthewidedynamicrangeofprotein
expressioninmusclefibers,whichspansseveralordersofmagnitude,fromthehighlyexpressed
contractileproteins,suchasmyosinandactin,tolowabundanceproteins,suchastranscription
factors.InourMS-basedshotgunproteomicsworkflow,wemeasuretheabundanceofindividual
peptidesafterelutioninananoflowliquidchromatographygradientandelectrosprayionization
intothemassspectrometer.Thesamepeptidesarenotalwayspickedforsequencingbythemass
spectrometer,makingdetectionpartiallystochastic.Becauseofthedramaticdifferenceinthe
expressionlevelofsarcomericproteinscomparedtotherestofthemuscleproteome,peptides
fromafewhighlyabundantproteinsaremoreoftenselectedforsequencing,thusinterferingwith
theidentificationoflessabundantproteins.Asaconsequence,theoverallnumberof
identificationsandthecoverageofcomparablyunder-representedproteins(suchastranscription
factors)arereduced.Unlikemuscle,othertissuesandcelllineshavealargefractionofproteinsof
similarabundancerangeand,therefore,thequantificationofalargefractionoftheirproteomeis
technicallyeasier.
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AwaytoaddressthisissueandidentifylowabundanceproteinsistocombinetheMSanalysisof
singlefiberswiththatofa“library”,consistingofadeepproteomeofwholemusclehomogenates,
extensivelyfractionatedtoreduceproteomecomplexity.Suchalibrarycontainssequence
informationforpeptidesthataredetectedbutnotsequencedinthesinglemusclefibers.Using
the‘matchbetweenruns’featureoftheMaxQuantanalysissoftwareitispossibletotransfer
peptideidentificationsfromthedeepmuscleproteome,whereagivenpeptideismuchmore
likelytohavebeenfragmentedandsequenced,toasinglefiber,whereoftenonlytheintact
peptidehasbeenmeasured(Deshmukhetal2015).Asaresult,thenumberofquantifiedproteins
inmusclefibersincreases,allowingtheanalysisofalargerfractionofthesinglemusclefiber
proteome(Murgiaetal.,2015).Thenumberofpeptideswhichareidentifiedusing‘match
betweenruns’withthehelpofa“library”isaboutdoublethoseidentifiedwithoutmatching.
Anadditionalissuetobeconsideredisthatsomemuscleproteinfamilies,suchasMYHs,consistof
isoformswithextremelysimilaraminoacidsequences.Therefore,thenumberofpeptideswhich
canbeusedtodistinguishtheisoformsisonlyafractionofthetotalpeptidenumbermeasuredfor
theseproteins.Asaconsequence,theprecisequantificationofeachisoformcanbechallenging
usingthealgorithmsusedfortheanalysisofMSdatainthestandardmode,whichattributes
commonpeptidestothe“proteingroup”withthelargestnumberoftotalpeptidesidentified.To
avoidtheriskofincorrectattributionofMYHpeptidestothedifferentisoforms,onlyunique
peptides,i.e.peptideswhicharepresentonlyinagivenisoform,mustbeusedforquantification
ofMYHs.
ThecorrectidentificationandquantificationoftheMYHisoformshasprovidedspecificmarkersto
definetheproteinprofileofthedifferentfibertypes.Inagreementwithpreviousimmuno-
histochemicalandbiochemical(SDS-PAGE)studies,proteomicanalysesalsoshowthatasignificant
numberoffibershaveahybridMYHcomposition.However,wefoundthatbothhumanand
murinemusclescontainahighpercentageoffiberswithalargelypredominantisoform(morethan
80%ofthetotal)andthuscanbesafelyattributedtoagiventype.Onecanthuscomparethefiber
typeprofileofrelativelypuremusclefibertypes.Inthefollowingsectionsofthisreviewwe
highlightthepowerofsingle-fiberproteomicstorevealnovelaspectsofmouseandhumanmuscle
biology.
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Fiber-type-specificdifferencesinmitochondrialproteins
Thediscoverypowerofsingle-fiberproteomicsisclearlyillustratedbythestudyofmitochondrial
proteins.Alargenumberofmitochondrialproteinswasdetectedinthesingle-fiberproteome
datasetsofthefourfibertypespresentinmouseskeletalmuscles(Murgiaetal.,2015).The
comparisonofthesedatasetsshowsthatmostmitochondrialproteinsaremoreabundantintype
2Aand2Xfiberscomparedtotype1fibers,while,notsurprisingly,theglycolytic2Bfibersshowed
thelowestvalues.Forexample,therelativeexpressionlevelsof43proteinsfromcomplexI,III,IV,
andV(ATPase),whichwererepresentedinallourselectedfibers,showedthefollowing
coefficientsofOXPHOXquantity:1(type2A),0.91(type2X),0.63(type1),and0.21(type2B)
(Schiaffinoetal.,2015).Asimilardistributionofrelativevalueswasseenfortheproteinsofthe
TCAcycle,withatendencyfortype2Xfiberstoshowthehighestvalues.However,isocitrate
dehydrogenase(IDH)showedauniquepatternoffibertypedistribution,differentfromthatof
othercomponentsoftheTCAcycle.
IDHisanenzymethatcatalyzestheoxidativedecarboxylationofisocitrate,producingα-
ketoglutarateandCO2inatwo-stepprocess,whichinvolvesoxidationofisocitrateto
oxalosuccinate,followedbythedecarboxylation,formingα-ketoglutarate.Inmammals,IDHexists
intwoisoforms:IDH3isatetramercomposedbytwoα subunits,oneβ subunitandoneγsubunit
andcatalyzestheoxidativedecarboxylationofisocitratetoα-ketoglutaratecoupledwiththe
reductionofNAD+toNADH,whereasIDH2isahomodimerandcatalyzesthesamereactionusing
NADP+asacofactorinsteadofNAD+.Singlemusclefiberproteomicshasshownthatthe
distributionofIDH2andIDH3variessignificantlyaccordingtofibertypeinmurineskeletalmuscles
(Fig.2A).Inparticular,IDH2levelsdonotcorrelatewithOXPHOSlevels,astheyarehighestintype
1fibers,slightlylowerin2Aandmuchlowerin2Xand2Bfibers.Incontrast,IDH3α,aswellas
IDH3γ,ismuchlessabundantintype1comparedto2Xfibers,thushasadistributionsimilarto
thatofotherTCAcycleproteins.
ThedifferentialdistributionofIDHisoformswasconfirmedbyimmunohistochemistry(Fig.2B).
TheantibodiestoIDH2giveastrongsignalintype1and2Afibers,weakerin2Xandweakestin2B
fibers,whereasantibodiestoIDH3αstainstrongly2Xand2Aandveryweaklytype1fibers.The
observationshavebeenrecentlyconfirmedcomparingslowandfast2Afibersinmousesoleus
(Langetal.,2018).Thisdifferentialdistributionlikelyhasanimportantfunctionalcounterpart,as
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IDH2supportsthereductionofNADP+toNADPHandthusprovidesareducedcofactorfor
glutathionesulphidereductaseandthioredoxinreductase,namelytheenzymesessentialfor
controllingthebufferingofsuperoxideviaglutathioneperoxidase,peroxiredoxinandsuperoxide
dismutase.TheabundanceofIDH2inslowfiberswouldthusimprovetheirabilitytocontrolredox
stateduringcontinuousmitochondrialATPgeneration(Fig.3).Thisinterpretationwasconfirmed
bythefindingthatnicotinamidenucleotidetranshydrogenase(NNT),theenzymethatcouplesthe
hydridetransferbetweenreducedNADHandNADP+toprotontranslocationacrosstheinner
mitochondrialmembrane,isalso10timesmoreabundantintype1fiberscomparedto2Xfibers,
inspiteofthegreaterabundanceofOXPHOSandmostothermitochondrialproteinsin2Xfibers
(Schiaffinoetal.,2015).IDH2andNNTarethemajormitochondrialenzymesinvolvedinNADPH
generation.Theirabundanceintheslowtype1fibersthussupportstheideathatthecontinuous
activityoftheslowfiberscausesagreaterROSproductionwhichiscounterbalancedbyagreater
abundanceofNADPHgeneratingenzymes.
Fiber-type-specificdifferencesintheadaptiveresponseofskeletalmuscles
ThediversityamongsinglemusclefibersisnotrestrictedtotheirspecializationinATP
regenerationortodifferentcontractileperformance.Thediversityinvolvesalsoregulatoryand
adaptiveprocesses.Thus,thesinglefiberanalysiscanrevealadaptivechangesthatmightremain
undetectedwhenmusclesampleswithheterogeneouscompositioninmusclefibersareanalysed.
Astrikingexamplecomesfromthestudyoffibertypespecificadaptationsduringagingofhuman
muscles(Murgiaetal.,2017).Singlefiberproteomicsconfirmedtheexpectedage-relateddecline
inmitochondrialenzymesforbothslowandfast2Afibers,butshowedunexpectedvariationsin
thecomplementofenzymesoftheglycolyticpathwayandofglycogensynthesisanddegradation.
Enzymesofglycolysisandglycogenmetabolismwerefoundupregulatedinslowmusclefibersof
elderlysubjects(average70years-old)comparedtoyoungsubjects(average24years-old),as
showninFig.4.Onecouldhypothesizethattheincreaseinglycolyticenzymesisacompensation
forthelossofmitochondrialfunction.Alternatively,itispossiblethatthemaineffectofthis
metabolicshiftisthecontrolofmusclefibertrophism.Inthisview,glycolyticintermediatescould
bedivertedtoprecursorsofnucleotides,aminoacids,andfattyacidstosustainmuscleprotein
synthesisinagedslowfibers.Thiscouldunderlietheobservedstrikingdifferenceinmusclefiber
trophismduringaging.Indeed,manypreviousstudiesshowthatthefastfibersofelderlysubjects
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aredistinctlyatrophic,whereasslowfiberscharacteristicallymaintaintheirsizeduringaging.(e.g.
LexellandTaylor1991;Callahanetal2014).Interestingly,theabovementionedisoformsofIDH
showadifferentbehaviourwithaging,characterizedbyadecreaseinIDH2inslowfibersand
stablevaluesifnotincreasedinfast2Afibers.
Otherinterestingexamplesofhowsinglefiberproteomicscanrevealchangesinopposite
directionsofdifferentfibertypescoexistinginthesamemusclearegiveninthestudyofmurine
soleusmuscledenervation(Langetal.,2018).Singlefiberquantitativeanalysisrevealedopposing
regulationofSERCA2(codedbyATP2A2)inslowandinfast2Afibers.Removalofneuronal
innervationtendstoconvertslowfibersintofasterfibersandfastfibersintoslowerfibers.Inthe
murinesoleusmuscle,theapproximatecompositionrecognizesa50%-50%proportionofslowand
fast(mainly2A)fibers.Thus,theimpactofdenervationonaveryabundantproteinasthe
sarcoplasmic/endoplasmicreticulumcalciumATPasesmightremainundetectedwhenwhole
musclesareanalyzed,andonlythesinglefiberquantitativeanalysiscanrevealopposing
regulationofSERCA2(codedbyATP2A2)inslowandinfast2Afibers(Langetal.,2018).
Conclusionsandperspectives
Thedistinctivefeatureofsinglemusclefiberproteomicsisthat,incontrasttothetraditional
immunohistochemicalandelectrophoreticmethodsfocusedonspecificproteincomponents,this
techniqueprovidesaglobalandunbiasedportraitofthewholemyofiberproteinprofile.Forthe
firsttimeitisthuspossibletocompare,withinthesamefiber,proteinsassociatedwithdifferent
cellstructures,fromtheplasmamembranetomitochondria,sarcoplasmicreticulum,myofibrils
andnucleus,aswellastherelativedistributionofenzymesinvolvedinvariousmetabolic
pathways.Otheromicapproaches,suchastranscriptomics,havealsobeenappliedtosingle
musclefibers(seeChemelloetal.,2011,2019),howeverchangesingeneexpressiondonot
alwaysreflectchangesinproteinabundance(seeAndersenetal.,1997,1999),thusonlyprotein
levelscanbeusedtodrawmeaningfulphysiologicalcorrelations(seealsoSchiaffinoetal.,2019).
Singlemusclefiberproteomicscanbeadiscoverytooltodefinethefunctionofknownmuscle
proteins,asillustratedbythefiber-type-specificvariationsintheIDHisoformprofilereferredto
above,andtoidentifynewfiber-type-specificproteins.Indeed,dozensofproteins,previouslynot
consideredinskeletalmusclestudies,appeartobeselectivelyexpressedinoneoranotherfiber
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type(seeTableS2inMurgiaetal.,2015,andTableS6inMurgiaetal.,2017).Proteomicdatathus
openanewwindowtoexploremusclefiberdiversity,bygeneratinghypothesesandsuggesting
experimentstovalidatethesehypotheses.Forexample,knockdownofIDH2,especiallywhen
combinedwiththelackofNNTthatoccursspontaneouslyincertainmousestrains(seeSchiaffino
etal.,2015),wouldbeexpectedtocauseincreasedROSproductioninslowtype1butnotinfast
type2fibersinresponsetoelectricalstimulationorforcedexercise(seeSchiaffinoetal,2019).
AlimitationofsinglemusclefiberproteomicsbasedonshotgunMSisthatitisunabletoresolve
alltheproteinvariantsderivedbyalternativesplicing.Top-downMS-basedproteomics,inwhich
intactproteinsareanalyzedratherthanpeptides,allowstobetterdistinguishproteinisoforms
derivedfromhomologousgenesandproteinsderivedbyalternativesplicingofthesamegene.
However,absolutequantificationofselectedproteinscanonlybeobtainedbytargeted
proteomics,addingpreciseamountsofspecificproteinslabeledbystableisotopestothesample
tobeexamined.Stableisotopeincorporationintroducesasmallmassdifferencetoidentical
peptidessothattheycanbedistinguishedbyMS.Differentmethodsforabsolutequantification
haveemergedoverthelastyearsincludingProteinEpitopeSignatureTags(PrESTs)(Zeileretal,
2012)andabsolutequantification(AQUA)(seeLindermannetal.,2017).Theyallrelyoneither
spikinginheavylabeledpeptidesorheavylabeledfulllengthproteins.
Differentproteomicapproachescanbealsobeusedtoanalyzethepost-translational
modificationsthatproteinsundergoinmusclefibers,asillustratedinarecentphosphoproteomics
studyontheeffectofexerciseinhumanskeletalmuscle(Hoffmanetal.,2015).Recent
methodologicaldevelopmentshaveshownthatsamplepreparationforphosphoproteomicscan
beconsiderablyscaleddownandmultiplexed,makingitamenabletotheanalysisofbiological
samplesofsmallsize(Humphreyetal.,2018).However,aphosphoproteomicsstudyofsingle
musclefibersisnotyetpossiblewithavailabletechniques,duetothetinyamountsofproteins
presentinsinglemusclefibers.Inconclusion,alikelyscenarioofthefuturedevelopmentof
musclefiberproteomicsisthatshotgunMSwillcontinuetoprovideaglobalpictureofthe
myofiberproteinprofileandwillbecomplementedbytop-downMSandtargetedproteomics
approachestodefineproteinvariantsgeneratedbyalternativesplicingandpost-translational
modifications.
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AfinalpointthatmustbekeptinmindisthatavailabledatabasesrequiredforMSsearchesdonot
includethelargeandcontinuouslyincreasingnumberofpreviouslyundetectedmicropeptides,
expressedfromlongnoncodingRNAs(lncRNAs)andcircularRNAs(circRNAs),manyofwhichhave
beenrecentlydiscoveredinskeletalandcardiacmuscle(MakarewichandOlson,2017;vanHeesch
etal.,2019).Thediscoveryofmicropeptidesfurtherincreasesthecomplexityofthe
“proteoforms”,i.e.themolecularformsofexpressedproteins,thatarepresentinanytissue,
includingskeletalmusclefibers(seeAebersoldetal.,2016).
Acknowledgements
WearegratefultoProf.MatthiasMannforhishelpandsupportinthegenerationofthe
proteomicdata.WealsothankDr.LeonardoNogaraforhishelpinthepreparationofthefigures.
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FigurelegendsFig.1.Graphicalillustrationoftheworkflowformassspectrometry-basedsinglemusclefiber
proteomics.Manualsinglefiberisolationandsnap-freezingisfollowedbyasinglebuffer/single
vesselprocedureforproteindigestion,followedbypeptidepurification.Liquidchromatography
connectedtonano-electrosprayprecedesMSanalysisofpeptides.Thecorrespondingspectrain
rawfilesareanalyzedusingthefreelyavailableMaxQuantcomputationalproteomicssoftware
platformandtherelatedPerseusframework.
Fig.2.Relativeabundanceofisocitratedehydrogenase2(IDH2)andIDH3αproteinsinthefour
fibertypespresentinmouseskeletalmuscle,asdeterminedbysingle-fiberproteomicsand
immunohistochemistry.A.Single-fiberproteomicsshowsthatIDH2hashighestlevelsintype1
andverylowlevelsintype2Xfibers;incontrast,IDH3αhashighestlevelsintype2Xandverylow
levelsintype1fibers.Type2AfibersshowrelativelyhighlevelsofbothIDH2andIDH3α,while2B
fibershavethelowestlevelsofbothisoformsduetotheirpoormitochondrialcontent. B.Fiber-
type-specificdistributionofIDH2andIDH3α intheslowsoleus(SOL)andfastplantaris(PLANT)
mouseskeletalmuscles,asdeterminedbyimmunohistochemistrywithspecificantibodies.Left
panels:Transversesectionswerestainedwithmonoclonalantibodiesspecificfortype1,2Aand
2Bmyosinheavychains(MYH)toidentifythedifferentfibertypes;type2Xfibersareunstained.
Middleandrightpanels:SerialsectionswerestainedforIDH2orIDH3αandco-stainedfor
dystrophin(green)tohighlighttheplasmamembrane.IDH2ismoreabundantintype1and2A
fibers,lessabundantin2Xandalmostundetectablein2Bfibers.Incontrast,IDH3αisexpressedat
muchhigherlevelsin2Xand2Afiberscomparedtotype1and2Bfibers.Thispatternof
expressioncloselycorrespondstotheMSvaluesderivedfromsingle-fiberproteomicsshowninA.
AdaptedfromSchiaffinoetal.,2015.
Fig.3.AlternativepathwaysoftheTCAcycleatthelevelofisocitratedehydrogenaserevealedby
single-fiberproteomics.Theyellowboxessuggestapossibleinterpretationtoaccountforthe
relativeroleofthetwopathways.TheNAD-dependentIDH3allowsthegenerationofmoreNADH,
thusmorefuelfortherespiratorychain(RC)andincreasedATPproduction,requiredbythemore
expensivemyosinmotorofthetype2Xfibers.TheNADP-dependentIDH2allowsthegenerationof
NDAPHrequiredbytheantioxidantmitochondrialsystemthatismoreabundantinthetype1
fibers.
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Fig.4.Age-dependentchangesinexpressionofglycolyticenzymesintype1and2Ahumanmuscle
fibers.Singlefibers,isolatedfromvastuslateralismusclebiopsiesofyounger(age22–27)and
older(age65–75)individuals,wereprocessedforsinglefiberproteomics.Thepercentagefold
change(older/youngerratio)isshownforeachproteininthetwofibertypes.Glycolyticenzymes
areindicatedbythecorrespondinggenenames.DatafromMurgiaetal.(2017).
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