DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young

13 Pages·2010·0.55 MB·English

JOURNAL OF PROTEOMICS 73 (2010) 1441–1453
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DIGE analysis of rat skeletal muscle proteins using nonionic
detergent phase extraction of young adult versus aged
gastrocnemius tissue
Pamela Donoghuea,1, Lisa Stauntonb,1, Edel Mullenb, Gwen Manninga, Kay Ohlendieckb,⁎
aConwayInstitute,UniversityCollegeDublin,Belfield,Dublin4,Ireland
bDepartmentofBiology,NationalUniversityofIreland,Maynooth,Co.Kildare,Ireland
A R T I C L E I N F O A B S T R A C T
Keywords: Contractileweaknessandlossofmusclemassarecriticalfeaturesoftheagingprocessin
Aging mammalians.Age-relatedfibrewastinghasaprofoundeffectonmusclemetabolism,fibre
Detergentphaseextraction typedistributionandtheoverallphysiologicalintegrityoftheneuromuscularsystem.This
DIGE studyhasusedmassspectrometry-basedproteomicstoinvestigatethefateoftheagingrat
Muscleproteomics muscleproteome.Usingnonionicdetergentphaseextraction,thisreportshowsthatthe
Sarcopenia aged gastrocnemius muscle exhibits a generally perturbed protein expression pattern in
boththedetergent-extractedfractionandtheaqueousproteincomplementfromsenescent
muscletissue.Inthedetergent-extractedfraction,theexpressionofATPsynthase,isocitrate
dehydrogenase,enolase,tropomyosinandbeta-actinwasincreased.Differentisoformsof
creatinekinaseandprohibitinshoweddifferentialchanges.Intheaqueousfraction,malate
dehydrogenase,sulfotransferase,triosephosphateisomerase,aldolase,cofilin-2andlactate
dehydrogenaseshowedincreasedlevels.Interestingly,differentialeffectsondissimilar2-D
spotsofthesameproteinspecieswereshownforCu/Znsuperoxidedismutase,albumin,
annexin A4 and phosphoglycolate phosphatase. Mitochondrial Hsp60, Hsp71 and
nucleoside diphosphate kinase B exhibited a reduced abundance in aged muscle. The
majority of altered proteins were found to be involved in mitochondrial metabolism,
glycolysis, metabolic transportation, regulatory processes, the cellular stress response,
detoxificationmechanismsandmusclecontraction.
©2010ElsevierB.V.Allrightsreserved.
1. Introduction metabolicalterationsintheagingorganism[3–5].Denervation
probably plays a key role in the age-dependent shift to a
Naturalagingisafundamentalbiologicalprocess[1],whereby slower-twitchingphenotype[6].Althoughlongitudinalstudies
oneofthemoststrikingfeaturesofthesenescentbodyisthe indicatethatcontractilefunctionmaybepreservedinsingle
lossinmusclemassandfunction[2].Age-relatedcontractile human muscle fibres [7], it has clearly been established
weakness has been termed sarcopenia of old age and is that significant alterations occur at the whole-muscle level
probably indirectly involved in many physiological and [8].Theage-relatedreductionincross-sectionalmusclearea
⁎ Correspondingauthor.Tel.:[phone];fax:[phone].
E-mailaddress:[email protected](K.Ohlendieck).
1 Bothauthorscontributedequallytothisbodyofwork.
1874-3919/$–seefrontmatter©2010ElsevierB.V.Allrightsreserved.
doi:10.1016/j.jprot.2010.01.014
1442 JOURNAL OF PROTEOMICS 73 (2010) 1441–1453
correlatesrelativelywellwiththedecreasedspecificforceof
2. Materials and methods
senescent muscles [9]. A variety of cross-sectional studies
agreethatthetissuemassoftheagedmusculaturedecreases
dramatically.Itisestimatedthat15to42%ofmusclemassis 2.1. Materials
lostbetweentheagesof20and80[10–12].
Severalmassspectrometry-basedproteomicstudieshave CyDye DIGE Fluor minimal dyes Cy3 and Cy5, ampholytes,
investigated changes in the protein complement of aging coverfluid,acetonitrile,thePlus-Onesilver-stainingkit,and
skeletalmusclesoverthelastfewyears,asreviewedbyDoran immobilised pH gradient (IPG) drystrips for 2-DE were
etal.[13].Theseproteomicprofilingstudieshavefocusedon obtained from Amersham Bioscience/GE Healthcare (Little
crudesolubleextractsandhaveshownthatmuscleagingis Chalfont,Bucks,UK).Sequencinggrade-modifiedtrypsinwas
associated with altered expression levels of many key purchased from Promega (Madison, WI, USA). LC–MS Chro-
contractile proteins, regulatory elements, ion handling pro- masolv water and formic acid were from Fluka (Milwaukee,
teins,metabolicenzymesandmediatorsofthecellularstress WI, USA). Acrylamide stock solutions were purchased from
response [14–20]. The proteomic finding of a drastically National Diagnostics (Atlanta, GA, USA). GE buffer systems,
decreaseddensityand reducedenzymaticactivity ofcertain proteinmolecularweightladdersandBradfordreagentwere
glycolytic enzymes in senescent fibres [21] is in agreement obtainedfromBioradLaboratories(Hemel-Hempstead,Herts.,
withtheideaofafast-to-slowtransformationprocessduring UK). Protease inhibitors were from Roche Diagnostics (Man-
aging [13]. In analogy, proteomic surveys clearly revealed nheim, Germany). Nitrocellulose transfer stacks were
increased levels of mitochondrial enzymes and metabolite obtainedfromInvitrogen(Carlsbad,CA,USA).Chemilumines-
transportersofaerobicmetabolisminagedmuscle[22].Thus, cence substrate was purchased from Roche Diagnostics
fibre shifting to slower twitch characteristics with a more (Mannheim, Germany). Primary and secondary antibodies
oxidative bioenergetic profile appears to occur in the aging wereobtainedfromvariouscommercialsources,aslistedin
neuromuscularsystem.Recently,comparativesubproteomics previous studies [16–18]. Ultrapure lysine for quenching the
ofthesolublemyofibril-enrichedfractionhasshowndistinct DIGE labeling reaction and all other analytical-grade chemi-
age-dependent effects on the contractile apparatus. The calswerepurchasedfromSigmaChemicalCompany(Dorset,
contractile protein exhibiting the most drastically changed UK).
expression was identified as the MLC2 isoform of the slow-
type myosin light chain [23], which also exhibits enhanced 2.2. Nonionicdetergentphaseextractionofmuscle
phosphorylationlevelsinagedfibres[24].Althoughfastand proteins
slowisoformsofmyosinheavychainswitchinthemajorityof
aged fibres, the dramatic increase in slow MLC2 expression Fractionationofcrudeskeletalmuscletissuepreparationsinto
was restricted to individual senescent cells [23]. Since post- an aqueous phase and a detergent-extracted phase was
translational modifications play a central role in protein carried out as previously described for cardiac muscle [27].
function and considerably increase protein diversity within Freshlydissectedgastrocnemiusmuscletissue(250mg)from
the skeletal muscle proteome, it is important to stress that 3 Wistar rats each of 3-month and 26-month of age were
glycosylation, tyrosine nitration, and tyrosine/threonine groundintoafinepowderunderliquidnitrogenusingapestle
phosphorylation are generally altered in senescent skeletal and mortar. The age groups studied represent young adult
muscles [21,24,25], thereby complicating global protein ex- animals versus senescent rats [16–18]. Ground tissue was
pressionanalysis. immediately added to 8ml of ice-cold phosphate buffered
Buildingontheproteomicfindingsfromtheabovedescribed saline(PBS;0.9%(w/v)NaCl,50mMsodiumphosphate,pH7.4)
surveys of total crude muscle extracts [13], this study has and2mlof10%(v/v)TritonX-114andleftovernightat4°Con
focusedontheproteomicprofilingofsenescentmuscletissue arotaryshaker.Thesuspendedsampleswerecentrifugedat
with respect to the aqueous versus the detergent-extracted 20,000g for 30min at 4°C to remove tissue debris. The
fraction.Nonionicdetergentphaseextractionwascarriedout supernatant was incubated for 30min at 37°C and then
with Triton X-114. This detergent is ideal for the phase centrifuged for 5000g at 25°C to separate detergent and
partitioningofsolubleandmembrane-associatedmusclepro- aqueous phases. The aqueous top layer was removed to a
teins,asfractionatedproteinsarenotdegradedorexcessively fresh 50mltube towhich 2ml of 10% (v/v) TritonX-114 was
denatured [26,27]. The method was previously validated in added.Thelowerdetergent-extractedphasewasresuspended
cardiac tissue and resulted in two distinct subproteomes by in8mlofice-coldPBS.Bothsampleswereplacedfor15minat
partitioning soluble and membrane-associated proteins into 37°C,followedbya15mincentrifugespinat5000gand25°C.
aqueous and detergent phases, respectively [27]. Here, 2-D Thisprocesswasrepeated3timesinordertowasheachfraction
fluorescenceDIGEanalysis[28]wasusedtocomparetheyoung andremovepotentialcross-contaminants.Differentlyseparat-
adultversusthesenescentratgastrocnemiusmuscle.Following ed proteins were extracted from each phase by acetone
the gel electrophoretic separation of the aqueous and the precipitationat−20°Cfor1h.Followingacentrifugationstep
detergent-extracted fraction from skeletal muscle homoge- at5000gfor30min,precipitatedproteinswereresuspendedin
nates, proteins of interest with a changed abundance were 1mlbufferand200µlofDIGE-compatiblelysisbuffer[18].The
identifiedbyMALDI-ToFMS,MALDI-ToF/ToFMSandESILC–MS/ total protein yield from starting material of 250mg of
MStechnology.Severalnewpotentialmarkersofmuscleaging skeletal muscle tissue was 5.16±0.38mg in the young AQ
wereidentifiedwhichmightbeusefulforthefutureestablish- phase(n=5),4.78±0.62mgintheoldAQphase(n=5),0.45±
mentofacomprehensivebiomarkersignatureofsarcopenia. 0.10mgintheyoungDTphase(n=5)and0.40±0.09mginthe
JOURNAL OF PROTEOMICS 73 (2010) 1441–1453 1443
old DT phase (n=5). Protein concentration was determined sion changes between young adult and aged samples from
bytheBradforddye-bindingassay[29]. the AQ and DT fractions were analyzed using Progenesis
SameSpots analysis software from Non Linear Dynamics
2.3. Gelelectrophoreticanalysis (NewcastleuponTyne,UK).Priortoanalysis,individualgels
werewarpedtoasinglemastergelandfurthermatchedand
Forthecomparativeproteomicanalysisofproteinsfractionated normalizedwithnon-gelspotsmanuallyfilteredoutonthe
intoAQphaseandDTphase,minimalfluorescentdyelabeling basisofspotvolume.SameSpotsanalysis(SSA)wasusedto
was performed at a ratio of 25µg of protein per 200pmol of identifyproteinsofinterestusingthefollowingparameters:
CyDyeaspreviouslydescribed[30],followingtherecommenda- n=5;ANOVAp<0.05;t-testp<0.05;andapowervalueof>0.8.
tionsofKarpandco-workersforoptimizedDIGEanalysis[31,32]. Proteins with a significantly changed abundance were
Labeledsampleswereincubatedonicefor30mininthedark. picked for tryptic digestion from silver-stained preparative
Thelabelingreactionwasterminatedbytheadditionof10mM gels.
lysine[18].Labeledproteinextractswereutilizedimmediately
for further analysis or otherwise stored at −80°C. Protein 2.5. Proteindigestion
samples,forseparationoverapH4–7gradient,werecombined
with an equal volumeof 2× lysisbuffer (9.5M urea, 2%(w/v) Excisedproteinspotsfrom2-DEgelswereaddedto200mM
CHAPS,2%(w/v)DTTand1.6%PharmalytepH3–10),followedby ammoniumbicarbonate solution andincubatedat37°Cfor
in-gelrehydrationovernightpriortoisoelectricfocusing.Afinal 10min with gentle agitation. Following a brief spin, a
strip volume of 450µl was achieved through the addition of solution of 200mM ammonium bicarbonate/acetonitrile 2:3
rehydrationbuffer(8Murea,0.5%(w/v)CHAPS,0.2%(w/v)DTT wasaddedtoeachgelplugandleftat37°Cfor10minwith
and 0.2% w/v Pharmalyte pH 3–10). First dimension gel gentle agitation. Samples were spun again briefly and
electrophoreticseparationwasperformedat0.05mA/IPGstrip resuspended in 50mM ammonium bicarbonate and left at
for75.000Vh,using8000Vfor10minand8000Vfor1hat20°C. 37°Cfor10minwithgentleagitation.Gelplugswereshrunk
BasicIPGphorstrips(18cmpH6–11)wereprehyratedin300µl using 100% acetonitrile for 10min at 37°C. Proteins were
basicrehydrationbuffer(8Murea,0.5%(w/v)CHAPS,0.2%(w/v) digestedovernightat37°Cwithgentleagitationusing0.2ng/
DTTand1.2%DeStreakReagent)overnightatroomtempera- μl of sequence grade-modified trypsin (Promega, Madison,
ture.Proteinsamples,forseparationoverapH6–11gradient, WI,USA).Peptideswererecoveredbyremovingsupernatants
werecombinedwithanequalvolumeof2×lysisbuffer(9.5M from digested gel plugs. Further recovery was achieved by
urea,2%(w/v)CHAPS,2%(w/v)DTTand1.2%DeStreakReagent). adding 30% acetonitrile/0.2% trifluoric acid to the gel plugs
Final sample volume was brought up to 100µl in basic re- for 10min at 37°C with gentle agitation. Following a brief
hydrationbufferandactivelypaperbridgeloadedontopH6–11 spin, supernatants were added to the initial peptide re-
IPGstripsfor16hat200V[33].Followingactivepaperbridge coveryfollowingdigestion.Exhaustivepeptiderecoverywas
sampleloading,allwickswerechanged.IEFwasperformedat achieved through the addition of 60% acetonitrile/0.2%
0.05mA/IPGstripat300Vfor2h,600Vfor2h,1000Vfor1h, trifluoric acid to each plug for 10min at 37°C with gentle
8000V for 1h and finally 8000V to achieve 50.000Vh. All agitation. Supernatants were added to the peptide pool.
focusedIPGstripswereimmediatelyusedforthe2Dseparation Sample volume was reduced until dry through vacuum
step or frozen and stored at −80°C. Strip equilibration was centrifugation. Sampleswere resuspended in 30μl of ultra-
carriedoutin10ml/stripofequilibrationbuffer(6M(w/v)urea, pureddH Oand0.1%formicacidforidentificationbymass
2
30%(v/v)glycerol,2%(w/v)SDS,0.05MTris–HCl,pH8.8and0.1% spectrometry.
(w/v)bromophenolblue)withtheadditionof1%(w/v)DTTfor
15min.Subsequently5%(w/v)iodoacetamidewasaddedtothe 2.6. ESILC–MS/MSanalysis
above equilibration buffer with strip incubation for a further
15min.2-Dseparationwascarriedoutat1W/gelforapprox24h ESI LC–MS/MS analysis was carried out in the Proteomics
using a Protean Plus Dodeca cell system (Biorad Laboratories Suite of the National University of Ireland, Maynooth.
(Hemel-Hempstead, Herts., UK) at 15°C and was terminated Nanoflow LC separation of tryptic peptides was performed
whenthedyefronthadjustmigratedoffthelowerendofthe with a nanoflow 1200 series system from Agilent Tech-
gels.Atotalof24gelswereanalyzedoverpH4–7andpH6–11 nologies (Santa Clara, CA, USA), equipped with a Zorbax
ranges.Thiscoveredbothyoungadultandagedgastrocnemius 300SB C18 5µm, 4mm 40nl pre-column and an Zorbax
specimenswithrespecttoAQ-andDT-phase-enrichedproteins. 300SB C18 5µm, 43mm×75µm analytical reversed phase
GelswerescannedusingaTyphoonvariablemodeimager9400 column using HPLC-Chip technology. Samples were initially
(Amersham Bioscience/GE Healthcare, Little Chalfont, Bucks, transferredwithanaqueous0.1%formicacidsolutiontoapre-
UK),withastandardpixelvolumeof60K–80Kforallscans.The column with a flow rate of 4µl/min for 3min. Mobile phase
isolationofcrudeextractsfromskeletalmuscletissue,1-Dgel A was 0.1%formicacidand mobilephase B 0.1% formic acid
electrophoresis,andsilverstainingof1Dor2Dgelswascarried in acetonitrile. Peptides were separated with a gradient of 3
outaspreviouslydescribedindetail[22]. to40%mobilephaseBover40minataflowrateof400µl/min,
followed by a 10min wash with 90% of mobile phase B. The
2.4. Expressionanalysis column was re-equilibrated at initial conditions for 10min.
Injection volume of 20µl was used per sample. Analysis of
Following nonionic detergent phase extraction of muscle trypticpeptideswasperformedusinganAgilent6300seriesion
proteins,DIGElabelingand2-DEseparation,proteinexpres- trapLCmassspectrometer.Allanalyseswereperformedusing
1444 JOURNAL OF PROTEOMICS 73 (2010) 1441–1453
positive nano-electrospray ion mode. The MS survey scan
3. Results
was set between 100 and 2200m/z with a duration of 0.5s/
scanandthe3mostintensepeptidesfurtherselectedforMS
analysis.Collisionenergywasrampedbetween1500and4500V 3.1. Nonionicdetergentphaseextractionfortheproteomic
basedontheobservedprecursormassoverchargevalueand analysisofagedskeletalmuscle
chargestate.SpectrawereprocessedandanalyzedbySpectrum
Mill database search software (Agilent Technologies, Santa Thesubproteomicinvestigationdescribedhereisbasedonthe
Barbara,CA). principle of nonionic detergent phase extraction [26]. A
comparison of the aqueous versus the detergent-extracted
2.7. MALDI-ToF/ToFMSanalysis fractionfromyoungadultversussenescentratgastrocnemius
muscle tissue was carried out. Fig. 1A outlines the experi-
MALDI-ToF MS analysis was performed in the Proteome mental protocol used for the separation of skeletal muscle
Research Centre of the Conway Institute, University College proteinsbytreatmentwithTritonX-114.Sincephasesepara-
Dublin. MALDI-ToF and MALDI-ToF/ToF mass spectrometric tion with Triton X-114 occurs at temperatures above 22°C,
analyses were carried out with a 4800 plus MALDI ToF/ToF skeletalmuscleproteinfractionationwascarriedoutat37°C
Analyzer (Applied Biosystems, Foster City, CA). Peptide [27]. The separation step resulted in an aqueous AQ phase-
masses were acquired over a range from 800 to 4000m/z, enriched in hydrophilic proteins and a detergent DT phase
withafocusmassof2000m/z.MSspectraweresummedfrom with predominantly hydrophobic proteins. Despite repeated
2500 laser shots from an Nd:YAG laser operating at 355nm washingsteps,phasetransitionapproachesalwaysresultina
and 200Hz. Calibration was performed using peptide stan- certain degree of cross-contamination between soluble and
dards (masses 900–2400m/z; Applied Biosystems). A maxi- membrane-associatedproteins.Extractedproteinsweresep-
mumof12precursorspersamplewellwithasignal-to-noise aratedbystandard2-DEandproteinsofinterestidentifiedby
ratio of >20 were automatically selected for subsequent MStechnology.
fragmentationbycollisioninduceddissociation.MS/MSspec-
tra were summed from 2000 laser shots. Spectra were 3.2. Gel electrophoretic analysis of nonionic detergent
processed and analyzed by the Global Protein Server Work- phaseextractionofmuscleproteins
station(AppliedBiosystems).
Fig. 1B–E illustrates the successful enrichment of typical
2.8. Immunoblotanalysis membrane-associated skeletal muscle proteinsin the deter-
gent-extractedfractionandasolubleenzymeintheaqueous
Immunodecorationofproteinsofinterestwascarriedoutby phase-extractedfraction.The1-DEanalysisofcrudeprepara-
1-Dimmunoblotanalysisaspreviouslydescribedindetail[17]. tions versus the aqueous phase versus the detergent-
Followingtheelectrophoretictransferofproteinstonitrocel- extracted phase demonstrated the differing protein band
lulose membranes, sheets were blocked in a milk protein pattern between the two main fractions following phase
solution for 1h and then incubated overnight with gentle separation(Fig.1B).Importantly, immunoblottingrevealeda
agitationwithprimaryantibody,sufficientlydilutedinblock- clear enrichment of the fast-twitch sarcoplasmic reticulum
ing solution containing 5% (w/v) fat-free milk powder in Ca2+-ATPase isoform SERCA1 (Fig. 1C) and the inner mito-
phosphate buffered saline (PBS; 0.9% (w/v) NaCl, 50mM chondrial membrane-associated enzyme ATP synthase
sodiumphosphate,pH7.4).Primaryantibodieswereobtained (Fig.1E)inthedetergent-extractedfractionfrombothyoung
fromAffinityBioreagents,Golden,CO,USA(mAbMA3-912to adult and aged muscle tissue. In stark contrast, the soluble
thefastSERCA1isoformofthesarcoplasmicreticulumCa2+- isoformofcytosolicmalatedehydrogenase(Fig.1D)wasfound
ATPase; and mAb MA3-913 to the fast CSQf isoform of to be enrichedin the aqueous phase from both young adult
calsequestrin), StressGen Bioreagents, Victoria, Canada (pAb andagedmuscletissue.Followingtheinitialevaluationofthe
SPA-801 to heat shock protein Hsp25) and Abcam Ltd., nonionic detergent phase extraction method by 1-DE, the
Cambridge, UK (pAb ab28172 to the mitochondrial marker aqueous and detergent fractions were electrophoretically
proteinprohibitin;pAbab48139tosuccinatedehydrogenase, separatedonlargehigh-resolution2-DEgelscoveringthepH
pAbab34830tomalate dehydrogenase;and mAbab14730 to 4–7andpH6–11rangeinthefirstdimension.Fig.2givesan
mitochondrial ATP synthase). Subsequently, blots were overviewoftheconsiderablydifferentexpressionpatternsof
washed twice with blocking solution for 10min before muscleproteinsderivedfromtheaqueousversusthedeter-
incubation for 1h with secondary peroxidase-conjugated gentphase.Sincesilverstainingexhibitsarelativelylimited
antibodies,dilutedinblockingsolution.Secondaryantibodies dynamic range, the more discriminatory DIGE method was
werepurchasedfromChemiconInternational(Temecula,CA, employedforadetaileddeterminationofchangesinprotein
USA).Followingfurtherwashingstepswithblockingsolution abundanceduetofibreaging.
and then two rinsing steps with PBS, antibody-decorated
bands were visualized by the enhanced chemiluminescence 3.3. DIGEanalysisoftheagedmuscleproteome
method following the manufacturer's recommendations
(Roche Diagnostics, Mannheim, Germany). Densitometric Fluorescent DIGE is one of the most powerful biochemical
scanning of immunoblots was performed on a Molecular tools for conducting comparative expression studies of
Dynamics300Scomputingdensitometer(Sunnyvale,CA,USA) complexproteomes,makingitanidealapproachforarefined
withImagequantV3.0software[22]. analysisofthedetergent-enrichedfractionfromyoungadult
JOURNAL OF PROTEOMICS 73 (2010) 1441–1453 1445
Fig.1–Nonionicdetergentphaseextractionofyoungadultversusoldmuscletissue.Thesubcellularfractionationapproach
usedinthissubproteomicinvestigationisbasedontheprincipleofnonionicdetergentphaseextraction.Theflowchartinpanel
(A)outlinestheexperimentalprotocolemployedfortheseparationofskeletalmuscleproteinsbytreatmentwithTritonX-114.
Thetemperature-dependentseparationstepresultedinanaqueous(AQ)phase-enrichedinhydrophilicproteinsanda
detergent(DT)phasewithpredominantlyhydrophobicproteins.Panel(B)showsasilver-stained1-DSDS-PAGEgelofmuscle
proteinfractions(100μgofprotein/gellane)followingnonionicdetergentphaseextraction.Lane1representsmolecularweight
markers(MW).Lanes2to7representcrudepreparationsfromyoungmuscle(YC),crudepreparationsfromoldmuscle(OC),
aqueousphase-enrichedproteinsfromyoungmuscle(YAQ),aqueousphase-enrichedproteinsfromoldmuscle(OAQ),
detergentphase-enrichedproteinsfromyoungmuscle(YDT)anddetergentphase-enrichedproteinsfromoldmuscle(ODT),
respectively.Panels(C)to(E)showcorrespondingimmunoblotslabeledwithantibodiestotheintegralSERCA1isoformofthe
sarcoplasmicreticulumCa2+-ATPase,thecytosolicisoformofmalatedehydrogenase(MDH)andthemitochondrialenzymeATP
synthase,respectively.30μgofproteinwasloadedpergellane.Thedistinctsubcellularfractionationoftheintegralversus
solublemarkerproteinsduringthedetergentphaseextractionproceduresagreeswiththeideaofasuccessfulseparationof
hydrophobicversushydrophilicmuscleproteins.
versusagedratgastrocnemiusmuscle.Itrepresentsahighly twodifferentIEFapproachesresultedintheseparationof657
accuratequantitativetechniquethatenablesmultipleprotein and371proteinspotsinthedetergentphase,and1464and373
samples to be separated in parallel on the same 2-DE gel, proteinspotsintheaqueousphase,respectively.Thenumber
therebygreatlyreducingtheintroductionofpotentialartifacts ofproteinsthatexhibitedachangedabundancewas23,15,37
dueto gel-to-gel variations [28]. Followingscanningwith an and19forthepH4–7DTphase,pH6–11DTphase,pH4–7AQ
AmershamTyphoonTriovariableimagerandwiththehelpof phaseandpH6–11AQphase,respectively.Proteinsofinterest
Progenesis 2-D analysis software, 94 protein species out of wereidentifiedbymassspectrometry.
2865 detectable 2-D spots were found to be differentially
expressed.DIGEmastergelsrepresentingelectrophoretically 3.4. Proteomicprofileofagedskeletalmuscle
separatedproteinsfromyoungadultversussenescentmuscle
fibresareshownforthepH4–7(Figs.3Aand4A)andthepH6– A list of DIGE-identified protein species with an altered
11(Figs.3Band4B)range.ThelowerandhigherpH-scaleofthe concentrationlevelintheDT-extractedandAQfractionfrom
1446 JOURNAL OF PROTEOMICS 73 (2010) 1441–1453
Fig.2–2-DEanalysisofyoungadultversusoldskeletalmuscle.Shownaresilver-stained2-DEgelsofthedetergent(DT;A,B)
andaqueous(AQ;C,D)phasefractionsfromyoungadult(A,C)andold(B,D)ratskeletalmuscle.Representativegelsareshown
forthepH4–7andpH6–11range.ThepHvaluesoftheIEFsystemandmolecularmassstandards(inkDa)ofthe2-DEgelsare
indicatedonthetopandontheleftofthepanels,respectively.
youngadultversusagedratgastrocnemiusmuscleisshownin actual and theoretical pI-value,andfoldchangeofindividual
Table1.Thetablesummarizesresultswithrespecttothetypeof proteinsaffectedduringmuscleaging.DatasetsfrombothpH4–
2-DE gel used for protein separation, the type of MS method 7andpH6–11gelsarecombinedinthelist.Thenumberingof
employed for protein identification, the protein accession distinct2-Dspotsofaltereddensityshowninthe4DIGECy5
number of identified muscle proteins, Mascot scores, the mastergelsofFigs.3and4correlateswiththe94MS-identified
number of matched peptide sequences, the percentage se- proteinspeciesinTable1.Overalladecreasedexpressionlevel
quencecoverage,theactualandtheoreticalmolecularmass,the wasshownfor35proteinspotsandanincreasedconcentration
JOURNAL OF PROTEOMICS 73 (2010) 1441–1453 1447
Fig.3–DIGEanalysisofthedetergent-extractedfractionfromyoungadultversusoldratskeletalmuscle.Shownare
Cy5-labeledmastergelsofthedetergent-extractedfractionfromratskeletalmuscle,coveringboththepH4–7(A)andpH6–11
(B)range.Proteinswithdifferentexpressionlevelsarenumbered1to38andaremarkedbycircles.SeeTable1foradetailed
listingofdetergent-extractedproteinswithachangedabundanceinsenescentfibres.ThepHvaluesoftheIEFsystemand
molecularmassstandards(inkDa)ofthe2-DEgelsareindicatedonthetopandontheleftofthepanels,respectively.
was found for 59 distinct protein species. Skeletal muscle dissimilar 2D spots representing the same protein or protein
proteins with an age-related change in abundance ranged in complexwereobservedformusclecreatinekinase,actinand
molecularmassfromapparent13.6kDato87.5kDaandcovered prohibitin. Several unidentified proteins showed a reduced
a pI-range from approximately 4.7 to 9.2. In the DT fraction, abundanceinagedmuscle.IntheAQfraction,proteinswithan
proteinspecieswithincreasedexpressionlevelswereidentified increased concentration were identified as cytosolic malate
as Coq7 protein, ATP synthase, isocitrate dehydrogenase, dehydrogenase,triosephosphateisomerase,bilesaltsulfotrans-
enolaseandtropomyosin.Interestingly,differentialeffectson ferase,PARK7 protein, HHIP-like protein 1,aldolase,pyruvate
Fig.4–DIGEanalysisoftheaqueousfractionfromyoungadultversusoldratskeletalmuscle.ShownareCy5-labeledmaster
gelsoftheaqueousfractionfromratskeletalmuscle,coveringboththepH4–7(A)andpH6–11(B)range.Proteinswithdifferent
expressionlevelsarenumbered39to94andmarkedbycircles.SeeTable1foradetailedlistingofaqueousphase-extracted
proteinswithachangedabundanceinsenescentfibres.ThepHvaluesoftheIEFsystemandmolecularmassstandards(inkDa)
ofthe2-DEgelsareindicatedonthetopandontheleftofthepanels,respectively.
1
4
4
Table 1–DIGE-identified proteins with a changed abundance in the detergent (DT)-extracted and aqueous (AQ) fraction from young adult versus old rat gastrocnemius 8
muscle.
Gel Spot Protein Nameofidentifiedprotein Mascot Peptides Coverage Actual Theoretical Actual Theoretical Fold ANOVA
no. accession score matched (%) molecular molecular isoelectric isoelectric change p<0.5
no. mass(kDa) mass(kDa) point(pI) point(pI)
DTpH4–7ESILC–MS/MS 1 gi|149068153| Demethyl-Q7(Coq7) 59 3 13 22.9 22.9 6.5 6.1 +1.3 0.037
2 gi|203055| ATPsynthase,alphasubunit 363 14 23 39.0 58.9 6.8 9.2 +1.5 0.032
3 – Unknownprotein – – – 22.0 – 7.3 – –1.7 0.009
4 gi|6978661| Musclecreatinekinase 82 5 14 43.2 43.2 7.2 6.6 −1.5 0.03
5 gi|6671762| Musclecreatinekinase 75 4 12 45.0 43.3 6.7 6.6 +1.9 0.019
6 gi|6679299| Prohibitin 337 18 74 45.0 29.9 6.0 5.6 +1.4 0.002
7 gi|6679299| Prohibitin 202 10 49 46.0 29.9 6.3 5.6 −2.4 0.033
8 gi|15778719| Tropomyosin,alpha 151 6 20 48.5 32.7 4.5 4.7 +1.2 0.028
9 gi|149049481| Enolase2,gammaisoform 85 4 13 48.5 33.3 6.3 5.2 +1.4 0.043
10 gi|6679299| Prohibitin 168 5 34 49.5 29.9 6.5 5.6 +2.1 0.037 JO
11 gi|16758446| Isocitratedehydrogenase3 343 11 31 50.0 40.1 6.2 6.4 +1.5 0.041 U
R
12 gi|149041704| Isocitratedehydrogenase3 350 17 36 49.5 39.4 6.3 5.9 +1.5 0.022 N
13 gi|16758446| Isocitratedehydrogenase3 369 11 36 50.5 40.1 6.3 6.5 +1.6 0.037 A
L
14 gi|126723393| Enolase3,beta 222 14 38 55.0 47.3 6.5 7.1 +1.3 0.045 O
15 gi|16758446| Isocitratedehydrogenase3 156 6 24 51.0 40.1 6.2 6.5 +1.2 0.016 F
16 gi|6978661| Musclecreatinekinase 82 6 14 57.0 43.2 6.2 6.6 +1.7 0.016 PR
17 gi|78365255| Dihydro-lipoamideS-acetyltransferase 160 11 21 67.7 67.7 5.9 8.8 +1.1 0.032 O
18 – Unknownprotein – – – 66.0 – 5.2 – –1.8 0.032 TE
19 – Unknownprotein – – – 51.0 – 4.9 – +1.4 0.033 O
M
20 gi|1374715| ATPsynthase,betasubunit 279 15 35 51.2 51.2 5.2 4.9 +1.4 0.028 I
C
21 gi|1374715| ATPsynthase,betasubunit 196 12 34 51.2 51.2 5.3 4.9 +1.4 0.011 S
22 gi|157823033| Actin,beta-2 76 6 15 51.0 42.3 5.4 5.3 +1.4 0.02 7
23 – Unknownprotein – 51.0 – 5.5 – +1.5 0.032 3(
DTpH6–11ESILC–MS/MS 24 gi|149043182| Actin,alpha-1 99 10 36 25.0 51.9 6.8 6.8 −4.7 0.002 20
25 – Unknownprotein – – – 49.0 – 6.2 – −2.4 0.033 1
0
26 – Unknownprotein – – – 23.0 – 7.6 – −2.4 0.046 )
1
27 gi|16758446| Isocitratedehydrogenase3 63 3 11 69.0 40.1 9.1 6.5 +2.4 0.044 4
28 – Unknownprotein – – – 62.0 – 6.5 – −2.4 0.009 41
29 – Unknownprotein – – – 68.0 – 6.6 – +2.4 0.02 –1
30 – Unknownprotein – – – 54.0 – 6.3 – −2.1 0.009 4
5
31 gi|6978661| Musclecreatinekinase 50 4 10 54.0 43.2 6.5 6.6 −1.9 0.043 3
32 – Unknownprotein – – – 49.0 – 6.5 – −1.6 0.006
33 – Unknownprotein – – – 57.0 – 8.8 – +1.4 0.04
34 gi|126723393| Enolase3,beta 276 24 40 68.5 47.3 7.3 7.1 +1.4 0.018
35 – Unknownprotein – – – 46.0 – 7.9 – −1.3 0.028
36 – Unknownprotein – – – 51.0 – 6.8 – −1.2 0.046
37 gi|126723393| Enolase3,beta 114 4 17 23.0 47.3 8.0 7.1 +2.5 0.074
38 – Unknownprotein – – – 70.0 – 8.1 – +2.4 0.058
AQpH4–7ESILC–MS/MS 39 – Unknownprotein – – – 44.0 – 6.3 – −1.4 0.039
40 – Unknownprotein – – – 25.5 – 5.6 – −1.7 0.017
41 – Unknownprotein – – – 29.0 – 5.5 – −1.4 0.038
42 gi|205474| Myosinlightchain 143 6 36 25.0 20.9 4.1 5.0 −1.7 0.03
43 gi|1213217| Cu/Znsuperoxidedismutase 158 6 47 27.5 16.2 6.9 5.7 −1.5 0.013
44 gi|203658| Cu–Znsuperoxidedismutase 150 4 32 42.0 15.9 6.8 5.9 +1.5 0.028
45 gi|16924002| Parkinsondiseaseprotein7 88 13 74 44.0 20.2 6.8 6.3 +1.5 0.017
46 – Unknownprotein – – – 46.0 – 6.9 – +2.0 0.045
47 gi|16924002| Parkinsondiseaseprotein7 116 6 56 45.0 20.2 4.6 6.3 −1.2 0.025
48 gi|9507245| Tyrosine3-monooxygenase/tryptophan 196 10 42 47.5 28.5 4.6 4.8 −1.5 0.017
5-monooxygenaseactivationprotein,
gammasubunit
49 gi|207349| Tropomyosin,alpha 261 19 49 62.0 32.7 4.1 4.7 +1.5 0.019
50 gi|55742832| AnnexinA4 282 10 35 51.0 36.2 5.5 5.4 +1.4 0.004
51 gi|37999910 AnnexinA4 161 6 26 32.0 36.2 4.0 5.3 −1.6 0.028
52 gi|38512111| TriosephosphateisomeraseTpi1 71 7 33 49.5 27.2 7.3 7.1 +1.3 0.048
53 gi|38512111| TriosephosphateisomeraseTpi1 141 8 52 48.0 27.2 7.4 7.1 +1.4 0.013
54 gi|38512111| TriosephosphateisomeraseTpi1 177 11 62 50.0 27.2 7.3 7.1 +1.2 0.042
55 giI38512111| TriosephosphateisomeraseTpi1 197 6 37 60.0 27.2 5.4 7.1 +1.4 0.011
56 gi|126723393| Enolase3,beta 417 19 40 70.0 47.3 6.6 7.1 +2.0 0.005
57 gi|126723393| Enolase3,beta 473 19 38 70.0 47.3 6.3 7.1 +2.1 0.005
58 gi|126723393| Enolase3,beta 284 10 28 68.5 47.3 5.1 7.1 −1.5 0.018
AQpH4–7MALDI-ToF/ToFMS 59 HSP7C_RAT HeatShockProteinHsp71 295 14 23 70.9 70.9 5.7 5.4 −2.7 0.003
60 ALBU_RAT SerumAlbumin 182 15 30 69.0 68.7 5.6 6.1 −2.2 0.016
61 ALBU_RAT SerumAlbumin 1070 23 48 69.0 68.7 5.8 6.1 +2.1 0.022
62 PGP_RAT Phosphoglycolatephosphatase 182 14 46 24.0 34.5 6.7 5.2 −1.4 0.039
J
63 ST2A1_RAT Bilesaltsulfotransferase1 180 3 17 42.0 33.2 6.6 7.7 +2.0 0.022 O
U
64 ENOB_RAT BetaEnolase 339 12 37 50.5 46.9 6.7 7.1 +1.8 0.043 R
65 HIPL1_RAT HHIP-likeprotein1 50 14 26 70.0 87.5 6.4 8.5 +1.4 0.035 N
A
66 PGP_RAT Phosphoglycolatephosphatase 125 7 26 43.0 34.5 5.6 5.2 +1.4 0.048 L
67 IDH3A_RAT Isocitratedehydrogenase(NAD)subunit 100 4 12 46.5 39.6 7.2 6.5 −1.8 0.010 O
alpha,mitochondrial F
P
68 ODP2_RAT Dihydrolipoyllysine-residue 158 9 18 67.0 67.1 6.9 8.8 −2.4 0.023 R
O
acetyltransferasepyruvatedehydrogenase T
69 IDH3A_RAT Isocitratedehydrogenase(NAD)subunit 363 10 26 47.0 39.6 7.0 6.5 +1.6 0.048 E
O
alpha,mitochondrial M
70 LDHB_RAT LactatedehydrogenaseBChain 59 10 26 44.0 36.6 6.1 5.7 +1.8 0.014 IC
71 Z354C_RAT ZincFingerProtein 62 9 23 67.0 64.2 7.3 8.6 −2.2 0.038 S
72 MDHC_RAT Malatedehydrogenase 64 2 8 46.5 36.5 6.8 6.2 +2.1 0.019 73
73 CSN4_RAT COP9signalosomecomplexsubunit4 279 9 31 49.0 46.3 5.7 5.6 −1.8 0.021 (
2
74 CH60_RAT 60kDaheatshockproteinmitochondrial 599 15 35 51.0 60.9 6.2 5.9 −2.6 0.003 0
75 ENOB_RAT Gammaenolase 169 11 40 49.0 47.1 5.4 5.0 −2.1 0.005 10
AQpH6–11ESILC–MS/MS 76 – Unknownprotein – – – 71.0 – 7.9 – +3.1 0.009 )1
77 gi|126723393| Enolase3,beta 314 15 36 49.0 47.3 6.1 7.1 +2.3 0.012 4
4
78 gi|55926145| NucleosidediphosphatekinaseB 77 7 51 26.0 17.4 7.2 6.9 −1.9 0.041 1
79 – Unknownprotein – – – 44.0 – 8.0 – +1.7 0.005 –1
4
80 gi|149041703| Isocitratedehydrogenase3 81 2 11 62.0 30.9 6.4 6.0 +1.7 0.016 5
81 – Unknownprotein – – – 21.0 – 8.8 – −1.6 0.013 3
82 gi|126723393| Enolase3,beta 122 7 20 51.0 47.3 8.9 7.1 +1.6 0.009
83 – Unknownprotein – – – 44.5 – 6.5 – +1.5 0.034
84 gi|16757994| Pyruvatekinase,muscle 130 8 20 61.0 58.3 7.5 6.6 +1.5 0.024
85 gi|109488172| Cofilin-2 90 2 25 57.0 13.6 6.1 8.9 +1.5 0.004
86 Unknownprotein – – – 22.0 – 6.7 – −1.5 0.028
87 gi|16757994| Pyruvatekinase,muscle 66 10 28 61.0 58.3 6.9 6.6 +1.5 0.024
88 gi|117935064| TriosephosphateisomeraseTpi1 195 8 41 61.0 27.4 7.1 6.9 +1.4 0.03
89 – Unknownprotein – – − 41.0 – 9.2 – +1.4 0.032
90 gi|6978491| Aldo-ketoreductasefamily1,B1 46 7 16 48.0 36.2 6.1 6.3 −1.4 0.005
91 gi|16757994| Pyruvatekinase,muscle 174 6 15 49.0 58.3 8.4 6.6 +1.3 0.041
92 gi|149067838| AldolaseA 49 4 10 50.0 36.4 9.2 8.5 +1.3 0.038
93 gi|38512111| TriosephosphateisomeraseTpi1 241 12 62 44.0 27.2 7.5 7.1 +1.2 0.043
94 gi|16757994| Pyruvatekinase,muscle 343 13 32 60.5 58.3 9.2 6.6 +1.2 0.026
1
4
4
9
1450 JOURNAL OF PROTEOMICS 73 (2010) 1441–1453
kinase,cofilin-2andlactatedehydrogenase.Differentialeffects validaterepresentativefindingsfromtheDIGEanalysisofyoung
ondissimilar2Dspotsofthesameproteinspecieswereshown adult versus aged muscle tissue. Western blotting revealed
for Cu/Zn superoxide dismutase, albumin, enolase, isocitrate comparablelevelsoftheperipheralproteinisoformCSQfofthe
dehydrogenase, annexin A4, and phosphoglycolate phospha- Ca2+-bindingprotein calsequestrinin theAQphase, aswell as
tase.COP9signalosomecomplexsubunit4,Hsp71,mitochon- similarlevelsoftheintegralCa2+-ATPaseandthestressprotein
drial Hsp60, tyrosine 3-monooxygenase/tryptophan 5- Hsp25 in the DT phase of 3-month versus 26-month old gas-
monooxygenaseactivationprotein,dihydrolipoyllysine-residue trocnemiusmuscle(Fig.5A–C).Thisdemonstratesequalloading
acetyltransferaseofthepyruvatedehydrogenasecomplex,zink oftheseunalteredproteinsduringtheelectrophoreticseparation
fingerprotein,nucleosidediphosphatekinaseB,myosinlight protocol.Incontrasttothesemuscleproteins,andinagreement
chainandaldo-ketoreductaseexhibitedareduceddensityin withtheDIGEanalysispresentedhereandinpreviousproteomic
agedmuscle.Themajorityofidentifiedproteinswerefoundto studies[13],ATPsynthase,succinatedehydrogenase,prohibitin
beinvolvedinmitochondrialmetabolism,glycolysis,metabolic andmalatedehydrogenaseweresignificantlyincreasedinaged
transportation, regulatory processes, the cellular stress re- preparations(Fig.5D–G).Thestatisticalevaluationisshownfor
sponse,detoxificationmechanismsandcontraction. age-related expression changes in ATP synthase, succinate
dehydrogenaseandmalatedehydrogenase(Fig.5H–J).
3.5. Immunoblotanalysisofage-relatedchangesinthe
skeletalmuscleproteome
4. Discussion
Basedontheaboveshownimmunoblottingofabundantmarkers
ofthesolubleanddetergent-extractedcohortofmuscleproteins The human genome is estimated to contain 25,000 genes of
(Fig. 1C–E), a similar immunodecoration analysis was used to which approximately 30% encode for membrane-associated
Fig.5–Immunoblotanalysisofthesubcellularfractionationoftheaqueousversusthedetergent-extractedfractionfrom
skeletalmuscletissue.Shownareexpandedviewsofimmuno-decoratedbandsofthefastCSQfisoformofcalsequestrin(A),
Ca2+-ATPaseisoformSERCA1(B),heatshockproteinHsp25(C),mitochondrialATPsynthase(D),succinatedehydrogenase(SDH)
(E),prohibitin(F)andmalatedehydrogenase(MDH)(G),respectively.Thedifferentfractionsanalyzedrepresentcrude
preparationsfromyoungmuscle(YC),crudepreparationsfromoldmuscle(OC),aqueousphase-enrichedproteinsfromyoung
muscle(YAQ),aqueousphase-enrichedproteinsfromoldmuscle(OAQ),detergentphase-enrichedproteinsfromyoungmuscle
(YDT)anddetergentphase-enrichedproteinsfromoldmuscle(ODT).Thegeneralproteinexpressionpatternoftheanalyzed
fractionscorrespondswiththefractionsshowninthesilver-stained1DgelofFig.1B.Significantchangesinabundancewere
foundformitochondrialATPsynthase,MDHandSDH,asdocumentedinthegraphicalpresentationofthestatisticalevaluation
ofantibodylabelinginpanels(H)to(J).ThecomparativeblottingwasstatisticallyevaluatedusinganunpairedStudent'st-test
(n=5;*p<0.05).Lanes1and2representproteinfractionsfrom3-monthversus26-montholdskeletalmusclefibres,
respectively.30μgofproteinwasloadedperlanepersampleforallimmunoblots.