Cytosolic proteomic alterations in the nucleus accumbens of cocaine overdose victims

19 Pages·2006·0.64 MB·English

MolecularPsychiatry(2007)12,55–73
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ORIGINAL ARTICLE
Cytosolic proteomic alterations in the nucleus accumbens
of cocaine overdose victims
N Tannu1, DC Mash2 and SE Hemby1
1DepartmentofPhysiology and Pharmacology,Wake ForestUniversity School ofMedicine,Winston-Salem, NC,USAand
2DepartmentofNeurology, University ofMiami School ofMedicine,Miami, FL,USA
Chroniccocaineuseinhumansandanimalmodelsisknowntoleadtopronouncedalterations
in neuronal function in the nucleus accumbens (NAc), a brain region associated with drug
reinforcement.Two-dimensionalgelelectrophoresiswasusedtocompareproteinalterations
in the NAc between cocaine overdose (COD) victims (n=10) and controls (n=10). Following
image normalization, spots with significantly differential image intensities (P<0.05) were
identified, excised, trypsin digested and analyzed by matrix-assisted laser desorption
ionization-time of flight-time of flight. A total of 1407 spots were found to be present in a
minimumoffivesubjectspergroupandtheintensityof18spotswasfoundtobedifferentially
abundantbetweenthegroups,leadingtopositiveidentificationof15proteinsbypeptidemass
fingerprinting (PMF). Ofan additional 37protein spotsthat were constitutively expressed, 32
proteins were positively identified by PMF. Increased proteins in COD included b-tubulin,
liprin-a3 and neuronal enolase, whereas decreased proteins included parvalbumin, ATP
synthaseb-chainandperoxiredoxin2.Thepresentdataprovideapreliminaryproteinprofileof
COD, suggesting the involvement of novel proteins and pathways in the expression of this
complex disease. Additional studies are warranted to further characterize alterations in the
differentially regulated proteins. Understanding the coordinated involvement of multiple
proteinsincocaineabuseprovidesinsightintothemolecularbasisofthediseaseandoffers
new targetsforpharmacotherapeutic intervention fordrugabuse-related disorders.
MolecularPsychiatry(2007)12,55–73.doi:10.1038/sj.mp.4001914;publishedonline31October2006
Keywords: human; post-mortem; mass spectrometry; 2-D fluorescence difference gel electro-
phoresis; synaptic signaling
Introduction of the NAc following imagery-induced drug
craving.6–8 These data support numerous studies in
Cocaine abuse/dependence is a devastating neuro- animal models demonstrating the role of the NAc in
psychiatric illness characterized by chronic and the reinforcing effects of cocaine.9–14
persistence drug use usually involving patterns of Although regional and neurotransmitter-centric
abstinence and binge use, impaired social and hypotheses have provided a framework for
occupational functioning, paranoid ideation, aggres- studying the acute and chronic effects of cocaine
sion, anxiety and depression.1 Historically, the abuse intake on the brain, detailed mechanisms of intra-
liabilityofcocaineisattributedtothedirecteffectsof cellular neuroadaptations have only recently
the drug on dopamine uptake blockade, yielding become known. Repeated cocaine use induces bio-
elevated extracellular dopamine concentrations that chemical adaptations in reinforcement-relevant
occur in discrete areas of the brain, specifically the brain regions15–17 and these adaptations appear to be
nucleus accumbens (NAc), ventral tegmental area relevant to the processes of sensitization, craving,
(VTA), prefrontal cortex – regions of the mesolimbic withdrawal and relapse.18 In humans, the functional
dopamine pathway, which originates in the VTA and activity of the NAc is associated with the sub-
projectstoseveralforebrainregions,mostnotablythe jective effects of cocaine6 and with craving for
NAc.2–5 In humans, neuroimaging studies have cocaine.7,8,19 In addition to the cocaine-induced NAc
revealed cocaine-induced functional activation of plasticity, studies in rodent models indicate persis-
the NAc following acute drug administration in tent or even permanent biochemical alterations in
cocaine-dependent subjects6 and bilateral activation regions associated with the mesolimbic dopamine
pathway including upregulation of the cyclic
Correspondence:DrSEHemby,MedicalCenterBoulevard,Wake AMP (cAMP) pathway,20–26 activator protein 1 family
Forest University-Bowman Gray School of Medicine, Winston- members27–32 as well as glutamate, dopamine, g-
Salem,NC27154,USA. aminobutyric acid (GABA) and opiate receptors,
E-mail:[email protected]
growth factors, cytoskeletal elements and cell
Received 26 May 2006; revised 1 August 2006; accepted 21
August2006;publishedonline31October2006 metabolism.33–41
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Genomic analyses in human post-mortem tissue of vided refined hypotheses regarding the molecular
cocaine overdose (COD) victims have confirmed the mechanisms of these diseases. Two-dimensional
findings of studies employing rodent models and electrophoresis (2DE), which separates protein spe-
have revealed novel mechanisms of drug-induced ciesbybothisoelectricpointandmolecularweight,52
neuronal and non-neuronal dysregulation in human has been widely used and recently been improved
post-mortem brain tissue.42–45 For example, Bannon through the addition of direct labeling of proteins
and co-workers42 examined gene expression in the with cyanine (Cy) dyes to facilitate quantitation
NAc of post-mortem brain tissue of human cocaine (two-dimensional difference gel electrophoresis
abusersandcontrolsandfoundsignificantalterations (2D-DIGE)).53–56 Quantitative proteomics, such as 2D-
in several novel functional classes and transcripts DIGE, is an open discovery platform (i.e. proteins are
including signal transduction, transcriptional and identified post hoc) in which proteins of interest are
translationalprocessing,neurotransmissionandsyna- excised from 2DE gels, digested with trypsin, ana-
ptic function, glia, structural and cell adhesion, lyzed by mass spectrometry and the masses are
receptors/transporters/ion channels, cell cycle and compared against predicted or known peptide pro-
growth and lipid and protein processing. Interest- files of proteins for matching via peptide mass
ingly, they identified several myelin-associated tran- fingerprinting(PMF).57–59Increasedaccuracyofidenti-
scriptsthatweresignificantlydecreasedintheNAcof fication can be accomplished through tandem mass
cocaine abusers including myelin basic protein spectrometry (MS/MS) to generate protein sequence
(MBP), proteolipid protein 1 (PLP) and myelin- data in addition to peptide masses.
associated oligodendrocyte basic protein (MOBP), The use of proteomic strategies, which provide
which were confirmed by quantitative PCR. In a broad and unbiased coverage of the proteome, can be
separate cohort, Hemby and colleagues evaluated a usedtounderstandthemultitudeofeffectsofcocaine
targeted set of transcripts in the VTA and lateral on protein function. In the present study, identifica-
substantia nigra (l-SN) and found significant upregu- tionofdifferentiallyexpressedproteinsfromtheNAc
lation of numerous transcripts and their encoded ofCODandcontrolswasassessedbyfluorescence2D-
proteinsintheVTA,butnotl-SN,ofcocaineoverdose DIGE combined with tandem mass spectrometry
victims including the N-methyl-D-asparatate receptor analysis. Protein expression profiles of COD and
subunitNR1,a-amino-3-hydroxy-5-methyl-4-isoxazole- controls were compared using DeCyder image ana-
propionic acid receptor subunit GluR2 and kainate lysis software and differentially expressed proteins
receptor subunits GluR5 and KA2 mRNAs.43 Simi- were identified by matrix-assisted laser desorption
larly, protein levels of NR1 and GluR2/3, with trends ionization-time of flight-time of flight (MALDI-ToF/
in GluR1 and GluR5, were found to be increased in ToF) mass spectrometry. In addition to proteins
the NAc but not putamen of cocaine overdose previously implicated in cocaine addiction, novel
victims,44 suggesting that these alterations are path- proteins were also identified.
way specific and may contribute to persistent drug
intake and craving in the human cocaine abuser.
Materials and methods
These studies suggest that multiple pathways are
involved in the neuropathology and altered neuronal Subjects and tissue
function of cocaine addicts. As noted for alcohol- Brain tissue was obtained at autopsy from age-
ism,46 such studies highlight the importance of using matched drug-free control individuals and COD
high-throughput technologies to study in more detail victims at the University of Miami Brain Endowment
the neuropathology associated with addictive dis- Bankasdescribedpreviously.60,61Adiagnosticneuro-
orders. Although these studies have been highly pathology examination at the gross and microscopic
informative in furthering our understanding of co- level, of multiple cortical and subcortical areas,
caine-induced transcriptional regulation contributing revealed no neuropathologicalabnormalities relevant
to long-term changes in primate brain, research to the mental status. The NAc from 10 control and
determining coordinate changes in the expression of 10 COD human subjects was used for this study
multiple proteins has been scarce.47 To develop a (Table 1). A semiquantitative neuropathological as-
more comprehensive understanding of the neuro- sessment found negligible neuronal loss, ischemic
adaptive processes involved in cocaine abuse, it is changes or reactive gliosis. All cases were retro-
necessary, reasonable and timely to evaluate regional spectively accrued based on the toxicological data
brain proteomes in human COD victims. To date, no andthecircumstancessurroundingdeathtocarefully
large-scale analyses of protein expression in the classify cocaine intoxication in the COD cases. This
human drug addicted brain have been made. included a review of the prior arrest records and
Recent technological developments have made the treatment admissions as well as pathological indica-
simultaneous, quantitative examination of hundreds tions such as perforation of nasal septum and needle
to thousands of proteins and protein modifications trackmarks.Allthecaseswereevaluatedforcommon
possible in post-mortem brain tissue of individuals drugs of abuse including alcohol. The positive urine
diagnosed with psychiatric illness, including alco- screens were confirmed by quantitative analysis of
holism.46,48–51 Such studies have confirmed the blood to exclude cases from the study based on
differential abundance of certain proteins and pro- evidence of polydrug or alcohol use before death.
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Table1 ClinicalcharacteristicsofCODandcontrolsubjects
Subjectno. Race/sex Age PMI(h) BrainpH Causeofdeath
CODsubjects
COD2 W/M 23 8 6.71 Cocaineintoxication
COD3 W/M 41 12 6.61 Cocaineintoxication
COD4 W/M 40 11.5 6.46 Cocaineintoxication
COD5 B/M 25 12 6.53 Cocaineintoxication
COD6 W/M 39 12 6.25 Cocaineintoxication
COD7 W/M 32 20 6.55 Cocaineintoxication
COD8 B/M 35 20.5 6.34 Cocaineintoxication
COD9 W/M 35 25.5 6.33 Cocaineintoxication
COD10 W/M 49 15 6.55 Cocaineintoxication
Mean 34.65 14.7 6.47
s.e.m. 3.11 1.84 0.05
Median 37 12 6.50
Controlsubjects
Control1 W/M 45 18 6.80 Bluntnecktrauma
Control2 W/M 27 14 6.42 Idiopathiccardiacconductionsystemdisease
Control3 W/M 44 11.5 6.29 Acutemyocardialinfarction
Control4 W/M 37 14.5 6.59 Atheroscleroticcardiovasculardisease
Control5 B/M 22 22 6.67 GSW
Control6 W/M 36 22 6.76 OCAD
Control7 W/M 34 14 6.29 Criticalcoronaryarteriosclerosis
Control8 B/M 33 15 6.62 Calcificaorticstenosis
Control9 W/M 34 11.5 6.63 Acuteasthmaticbronchitis
Control10 W/M 46 11 6.43 Aorticaneurysm
Mean 36 15.35 6.55
s.e.m. 2.56 1.35 0.06
Median 35.5 14.25 6.61
Abbreviation:COD,cocaineoverdose.
Cocaine and benzoylecgonine concentrations were and1mg/mlpepstatin.Homogenateswerecentrifuged
assessed by gas chromatography–mass spectrometry using a swinging bucket rotor (Beckman Coulter
from the brain as well as blood.62 SW55Ti) at 5333g for 5min and the supernatant
(cytosolic and crude membrane fraction) fractions
Protein isolation and fractionation were further centrifuged at 59255g for 30min at 41C
and the cytosolic supernatant fraction collected was
The brains were photographed after removal and cut
stored at (cid:1)801C. The cytosolic protein fraction from
into 1.5cm coronal blocks to be subsequently frozen
in 2-methyl butane on dry ice at (cid:1)301C and stored each subject was precipitated with 2D clean-up kits
later at (cid:1)801C. One hundred and fifty milligram (GEHealthcare,Piscataway,NJ,USA;accordingtothe
manufacturer’s recommendation) at (cid:1)201C overnight.
punches were dissected from the blocks containing
Thenextday,sampleswerepelletedbycentrifugation
NAc and caudate/putamen. The frozen brain tissue
at 12000r.p.m. for 5min. The pellets were air-dried
washomogenizedintoadryhomogenateusingasteel
for 2min and resuspended in 50ml of sample buffer
mortar and pestle chilled in dry ice. Subcellular
fractionation into membrane, nuclear and cytoplas- (30mM Tris-HCl, 2M thiourea, 7M urea and 4%
CHAPS, pH 8.5). After the samples were brought
mic fractions was performed as described pre-
viously.43,44 For the purposes of this study, only within a pH range of 8.0–9.0 using 0.1N NaOH, the
protein concentration was determined using the 2D-
cytosolicfractionswereevaluated.Studiesareunder-
Quant kits (GE Healthcare).
way to assess the membrane and nuclear proteomes
using alternative separation methodologies to enable
detection of hydrophobic proteins difficult to detect Cyanine dye labeling
by gel-based methods. Tissue samples were homo- Minimal labeling ofthe lysineresidues was achieved
genizedin10mMHEPES,10mMNaCl,1mMKH PO , by reaction with Cy dyes. A normalization (pooled)
2 4
5mM NaHCO , 1mM CaCl , 0.5mM MgCl , 5mM samplewaspreparedbycombining50mgfromeachof
3 2 2
EDTA, 1mM phenylmethylsulfonylfluoride, 10mM the 20 samples. The labeling of 50mg of protein
benzamidine, 10mg/ml aprotinin, 10mg/ml leupeptin sample(eachsample)wasoptimizedbylabelingwith
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200pmol of appropriate dye (suspended in >99.5% dithiothreitol (DTT), 4% CHAPS and 2% pharmalyte
pure dimethylformamide).63Thispermitted optimum (GEHealthCare))wasaddedandthetotalvolumewas
labeling of the comparatively less abundant protein brought up to 450ml by Destreak rehydration buffer
spots while still maintaining the most abundant (GE HealthCare).65 For the ‘pick gel’, 50mg of pooled
protein spots in the dynamic linear range for sample was labeled with the Cy2 dye, which was
quantitative image analyses. Each gel comprised a combined with 750mg of unlabeled pool sample. An
pooled sample labeled with Cy2 as well as COD and equal amount of sample buffer was added and the
control samples labeled with either Cy3 or Cy5 as total volume increased to 450ml by the addition of
depicted in Figure 1 and outlined in Table 2. The Destreak rehydration buffer.
labeling reaction was stopped by addition of 1ml of
10mM lysine on ice for 10min. For each gel, the Two-dimensional polyacrylamide gel electrophoresis
labeled pool sample and the labeled COD and The isoelectric focusing (IEF) was carried out on
control samples were mixed and an equal amount Immobiline DryStrips (240(cid:2)3(cid:2)0.5mm, linear 4–7
of rehydration buffer (2M thiourea, 7M urea, 2% pH,GEHealthcare,Piscataway,NJ,USA)usingtheGE
Figure 1 Schematic of 2D-DIGE procedure comparing cytosolic fractions from COD and control subjects. The schematic
represents comparison ofa COD victimandanage-matched controllabeledwith Cy3dyeandCy5dye,respectively.Note
that dye labeling was counterbalanced across groups for the study. A normalization pool comprising all COD and control
samples was labeled with Cy2 dye and used to normalize spot location across all gels. Following labeling, samples were
mixedandelectrophoresedbytwo-dimensionalpolyacrylamidegelelectrophoresis(2D-PAGE).Tensubjectsfromeachgroup
wereelectrophoresedonten2Dgelssimultaneously.Eachgelwasscannedatthreewavelengths(l)basedontheexcitation
wavelengthsofeachdye.TheimageswerefurtheranalyzedbyDeCyderImageanalysissoftwaretodeterminedifferentially
regulatedproteinspots.Gelimagesarerepresentativeanddepictadefinedsectionofthegel.
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Table2 Experimentaldesign (633nm) for excitation and 670 band pass emission
filter was used for scanning Cy5-labeled sample. The
Gel Cyaninedye2 Cyaninedye3 Cyaninedye5 photomultiplier tube was set to ensure maximum
no. Subjectcode
pixel intensity of 85000–95000 for all the images in
every gel. To remove the extraneous areas of the gel
1 Normalizationpool COD1 Control1 image, the images were cropped using the Image-
2 Normalizationpool Control2 COD2
Quant V5.2 (GE Healthcare). The image analysis was
3 Normalizationpool COD3 Control3
performed by DeCyder 5.01 (GE Healthcare). The
4 Normalizationpool Control4 COD4
DeCyder DIA was initially employed for intra-gel
5 Normalizationpool COD5 Control5
analysis, followed by DeCyder BVA for inter-gel
6 Normalizationpool Control6 COD6
7 Normalizationpool COD7 Control7 analysis. The DIA mode of DeCyder was employed
8 Normalizationpool Control8 COD8 forproteinspotdetectionaswellasfornormalization
9 Normalizationpool COD9 Control9 of COD and control gel images to the pooled sample
10 Normalizationpool Control10 COD10 gel image. After spot detection, the abundance
changes are represented by the normalized volume
Gel11:Normalizationpool(200mg)–preparativegel. ratio(Cy3:Cy2and Cy5:Cy2), representedbytheratio
of COD or the control sample (Cy3 or Cy5) with
respecttothepooledsample(Cy2).TheDeCyderDIA
normalizationmethodassumesthatmostproteinsare
Healthcare EttanIPGphor apparatus.64,65 Sequential unchanged, having unit volume ratio. The following
IEF was done by step-and-hold as follows: 10h at parameters were used for spot filtering: slope >1.0,
0V, 1h at 100V, 1h at 500V, 1h at 1000V, 3h at area <350, peak height <350 and volume <100000.
8000V (gradient;13.5kVh),7.5hat8000Vfora total The spots were authenticated manually for all the
of 75.1kVh. A maximum current of 50mA was gels.Spotmapsfromallthegelswerefirstmatchedby
applied per strip, maintaining the platform tempera- manual landmarks and then in automatic mode by
ture at 201C. After IEF, the IPG strips were equili- DeCyder BVA. The protein spot matches were con-
brated to reduce the disulfide bonds with gentle firmed manually for all the gels. The average ratio
rockingfor10mininequilibratingsolution(6Murea, (COD:control (n=10)) as well as the corresponding
1.5M Tris-HCl, pH 8.8, 30% (v/v) glycerol, 2% (w/v) Student’s t-test value for each protein spot was
sodiumdodecylsulfate(SDS)and2%(w/v)DTT).To calculated based on all gel images in the DeCyder
alkylate the SH groups of the proteins, the IPG strips BVA mode.50 The protein of interest based on
were rocked for 10min in solution containing 6M statistically significant t-test was further assessed by
urea, 1.5M Tris-HCl, pH 8.8, 30% (v/v) glycerol, thethree-dimensionalspotprofiletovalidatethespot
2% (w/v) SDS and 2.5% (w/v) iodoacetamide.66 The on most of the gel images.
proteins were further separated on the basis of their
molecular weight on a pre-cast 8–15% gradient SDS- Mass spectrometry analysis
polyacrylamide gel electrophoresis (SDS-PAGE) Protein spots of interest were subjected to mass
(2400(cid:2)2000(cid:2)1mm, Jule Inc., Milford, CT, USA) at spectrometry analysis for identification.70 The pick
a constant 4W using the Ettan Dalt II System (GE list of protein spots of interest was created using
Healthcare).67 Ten microliter of the molecular mass DeCyder DIA.71 Gel plugs of 2mm size were cut
marker (Amersham Rainbow marker RPN 800) was robotically using the Ettan Spot Handling Work-
loaded on 2mm2 filter paper, which was placed on station (GE Healthcare).50 Picked proteins were pre-
the basic end of the IPG strip.67,68 The slab gels were paredformassspectrometryanalysisusingastandard
fixedovernightin30%(v/v)methanoland7.5%(v/v) in-geltrypsindigestionprotocol.Briefly,thegelplugs
acetic acid in distilled water.69 This was followed by were washed for 20min, twice in 50mM ammonium
washing the gels twice with DW (500ml) for 10min bicarbonate/50% (v/v) methanol in water and once
each. The ‘pick gel’ after fixation was stained with with75%(v/v)acetonitrile(ACN)inDWfor30minor
SyproRuby (Molecular Probes, Eugene, OR, USA) until the gel plugs turn opaque. The ACN was
overnight as per the recommendations of the manu- removed after the final wash and dry gel plugs were
facturer. The next day, excess stain was removed by dried at 401C for 10min. After the samples were
washing the gel twice with 10% methanol and 6% cooled, they were incubated at 561C for 45min in
glacial acetic acid for 10min each. 40ml of 50mM DTT/100mM ammonium bicarbonate.
Thesampleswereallowedtocoolafterdiscardingthe
Gel image analysis solution and immediately 40ml of 100mM iodoacet-
TheTyphoon9400scanner(GEHealthcare)wasused amide was added and the samples were incubated at
to scan all the gels at 100mm resolution. The blue room temperature (RT) for 30min in dark. After
laser (488nm) for excitation and 520 band pass discardingtheabovesolution,afinalwashwasgiven
emission filter was used for scanning the gel image by100mlof50mMammoniumbicarbonate/50%(v/v)
of Cy2-labeled sample, the green laser (nm) for methanol in water for 10–15min at RT followed by
excitation and band pass emission filter was used drying the gel plugs by vacuum centrifugation.
for scanning Cy3-labeled sample and the red laser Twenty micrograms of lyophilized trypsin (883pmol;
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Promega, Madison, WI, USA) was reconstituted in following parameters were used: one allowed missed
1ml of 20mM ammonium bicarbonate and incubated cleavage, 750p.p.m. for mass/charge (m/z) error for
for 15min at 371C. After drying, the gel plugs were massspectrometryand0.1Damass/charge(m/z)error
incubated overnight at 371C in 140ng of sequencing for MS/MS, partial modification of cysteine (carba-
grade trypsin (Promega). The supernatant from tryp- midomethyl-cysteine) and methionine (oxidized).
sin digest was transferred to a low-retention 96-well PMF and MS/MS spectra were interpreted with the
plate. Peptides from the gel pieces were sequentially Mascot software (Matrix Science Ltd, London,
extracted twice in 100ml of extraction buffer (50% UK).73,74 Database searches, through Mascot, using
(v/v)ACNand0.1%(v/v)trifluoroaceticacid(TFA)in combinedPMFandMS/MSdatasetswereperformed
DW). The original tryptic supernatant and the super- viaGPSExplorersoftware(AppliedBiosystems).The
natants from two sequential extractions were com- probabilityscorecalculatedbythesoftwarewasused
bined and dried in a vacuum centrifuge. The dried as the criterion for correct identification. The algo-
peptides from each gel plug were dissolved in 5ml of rithm used for determining the probability-based
50% (v/v) ACN and 0.1% TFA in DW and 0.5ml scoring with a given mass spectrum is described in
deposited on the stainless-steel MALDI target plate. detail by Pappin et al.75 The criterion for identifica-
Afterdrying,thiswasmixedwith0.5mlof6mg/mlof tion was a MASCOTconfidence interval greater than
a-cyano-4-hydroxy-cinnamic acid (Sigma-Aldrich, 95%. Protein identification was confirmed by check-
St Louis, MO, USA) in 50% (v/v) ACN and 0.1% ing the protein mass and pI accuracy.70
TFA in DW.
Mass spectrometry analyses were performed using
the Applied Biosystems 4700 Proteomics Analyzer
Results
(MALDI-ToF/ToF: Applied Biosystems, Foster City,
CA,USA)inreflectormodeforpositiveiondetection.
Cytosolic fractions of NAc from human COD victims
The laser wavelength and the repetition rate were
and controls were compared using the DIGE proteo-
355nm and 200Hz, respectively. All the mass
mics strategy to determine differences in the abun-
spectrometry spectra resulted from accumulation of
dance levels of proteins in a pH range of 4–7 to
at least 2000 laser shots. The peak detection criteria
elucidate the molecular mechanisms by which pre-
used were minimum S/N of 8, local noise window
vious exposure to cocaine alters the cytosolic pro-
width (m/z) of 200 and minimum peak width at full-
teome of NAc (Figure 1). The total number of protein
width half-maximum (bins) of 2.9. The mass spectra
spots detected in the NAc of control and COD were
were calibrated using the two trypsin auto-digest
1555. Of these, 1407 protein spots were present in at
products: fragment 100–107 ([MþH]þ=842.51Da),
least five or more control and COD subjects. The
fragment 98–107 ([MþH]þ=1045.556Da) and frag-
DeCyder image analysis of the images representing
ment 50–69 ([MþH]þ=2211.105Da).72 Mass lists
COD and control NAc cytosol proteome elucidated
weresubmittedtoGPSExplorer(AppliedBiosystems)
the putative COD-specific protein spots. Quantifica-
for protein identification.57 A maximum of 10 stron-
tion of the individual protein spots revealed that the
gest precursor ions per sample were chosen for MS/ vast majority of B1400 protein spots were quantita-
MS analysis. The following monoisotopic precursor
tively similar. As anticipated, the majority of these
selection was used for the MS/MS: minimum S/N
proteins were gene products relegated to structural
filter of 10, excluding the most commonly observed
andhousekeepingfunctions.Imageanalysisoffluore-
peptide peaks for trypsin and keratin, and excluding
scently labeled NAc cytosol lysates loaded together
the precursors within 150 resolution. In the ToF1
on 2D gels identified differential distributions of 18
stage, all ions were accelerated to 1kV under condi-
protein spots characterized by 15 proteins after
tions promoting metastable fragmentation. The peak
chronic exposure to cocaine (Figure 2). The most
detection criteria used were S/N of 8 and local noise
prominent categories of altered expression were
window width of 250 (m/z).
membrane-associated proteins and proteins involved
in cytoskeletal transformation. Even though there
Protein identification have been many studies reporting the cocaine-
Mass lists were submitted to GPS Explorer (Applied induced alterations in gene expression, this is the
Biosystems) to search against the NCBInr (National first study that utilizes a non-candidate approach to
CentreforBiotechnologyInformationnon-redundant) decipher the involvement of novel proteins and
primate database for protein identification.57 The pathways.
Figure 2 Representative 2D-DIGE-based comparison of control and COD groups. A representative 2D-DIGE image of
cytosolicproteinsfromhumantheNAccontrolgroup.Cytosolicproteinswerefractionatedinthefirstdimensionbya4–7
linearpHgradient(pI)andintheseconddimensionbyan8–15%gradientSDS-PAGEgel(molecularweight,kDa).Theareas
markedwithboxesaremagnifiedandshownseparately(A–G)andarecomparedwiththecorrespondingareasfromtheCOD
group (A0–G0). The numbered protein spots marked with arrows are differentially regulated between control and COD
subjectsandcorrespondwithnumbersinFigures2and3,andTable3.
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Figure3 DifferentiallyexpressedproteinsbetweenCODandcontrolgroups.Quantitativeassessmentsofproteinspotswere
made using DeCyder-based quantitation software by comparing Cy2-normalized signal intensities for COD and control
subjects.Eachpaneldepictstheage-matchedCOD/controlpairs(filledcircles)andmeanstandardizedlogabundanceofthe
groups (red-filled star). Three-dimensional images of the identified protein spots from representative COD and control
subjectsareprovidedandthestandardizedlogabundancevaluefortherespectiveoutlinedpeaksareidentifiedastheblue
circlesandlinesintheplot.Allproteinsinthefigurearesignificantlydifferentbetweenthetwogroups(P<0.05).
Several proteins exhibited statistically significant proteins were statistically decreased in the COD
increases in COD victims including tubulin beta 2 victims including ATP synthase b-chain (ATPB),
(TUB2), desmoplakin, 60kDa chaperone, liprin-a3 peroxiredoxin 2, isoform a (PRDX2) and centrosome
and neuron-specific enolase (NSE), whereas other protein cep290 (Figures 3 and 4). Several protein
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Figure4 DifferentiallyexpressedproteinsbetweenCODandcontrolgroups.Quantitativeassessmentsofproteinspotswere
made using DeCyder-based quantitation software by comparing Cy2-normalized signal intensities for COD and control
subjects.Eachpaneldepictstheage-matchedCOD/controlpairs(filledcircles)andmeanstandardizedlogabundanceofthe
groups (red-filled star). Three-dimensional images of the identified protein spots from representative COD and control
subjectsareprovidedandthestandardizedlogabundancevaluefortherespectivepeaksareidentifiedasthebluecirclesand
linesintheplot.Allproteinsinthefigurearesignificantlydifferentbetweenthetwogroups(P<0.05).
spots showed a strong trend towards differential unique proteins. The magnitude of differential ex-
expression, but were not statistically significantly pression varied from a 1.21-fold decrease for brain-
different(spot2:glialfibrillaryacidicprotein(GFAP) type aldolase (spot 15;Figures 2 and 4) to a 3.27-fold
(P=0.084);spot5:sodiumchannelassociatedprotein increase in the steady-state levels of TUB2 (spot 4;
1 (P=0.079); and spot 15: brain type aldolase Figures 2 and 3). Notably, the expression of GFAP
(P=0.067)); however, the magnitude of differential (spots 1 and 2) was greater in COD victims as shown
expression warrants their inclusion. A representative in Figures 2 and 3.
DIGEgelimageofthecontrolNAccytosolisshownin
Figure 2. The areas of interest are magnified to show Methodological considerations
the abundance patterns of 18 protein spots, which Many of the proteins identified were confirmed by
were differentially regulated and characterized as 15 sequencing the peptide mass spectra obtained by
MolecularPsychiatry
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Table3 DifferentiallyexpressedproteinsfromhumanNAcofcontrolandCOD,identifiedbyMALDI-ToF/ToF
Spotno. ProteinID(PMF) Accessionno. M (kDa) pI Peptides MASCOT Confidence Proteinquantitation
r
score interval(%)
t-Test Averageratio
Structural
838 Glialfibrillaryacidicprotein gi|4503979 49.84 5.42 30 262 100 0.055 1.60
641 Glialfibrillaryacidicprotein gi|4503979 49.84 5.42 18 55 95.47 0.084 1.41
339 Tubulin,beta,2 gi|20809886 49.92 4.78 16 113 100 0.0039 2.72
388 Tubulin,beta,2 gi|20809886 49.92 4.78 14 74 99.9 0.004 3.27
P
1154 Dpeemsmpohpiglauksinan(tDigPe)n()250/210kDaparaneoplastic gi|12644130 33.16 6.44 59 62 99.9 0.012 1.34 roteom
376 60kDachaperonin gi|41399285 61.01 5.7 20 57 99.8 0.022 1.41 ics
a
n
d
S15y0napticplastiScoitdyiumchannelassociatedprotein1 gi|24308518 80.27 5.98 23 61 99.7 0.079 (cid:1)1.45 NTancocain
ne
386 Liprin-a3 gi|32189362 133.41 5.53 30 55 99.7 0.0072 2.56 uetabu
alse
Mitochondrial
640 Mitochondrialcarnitine/acylcarnitine gi|3914023 32.92 9.48 11 51 86 0.079 (cid:1)1.47
carrierprotein(CAC)
517 ATPsynthaseb-chain,mitochondrialprecursor gi|114549 56.52 5.26 18 78 99.97 0.036 (cid:1)1.31
Metabolism
688 Fructose-bisphosphate aldolaseC gi|113613 39.43 6.41 16 74 99.94 0.067 (cid:1)1.21
(brain-typealdolase)
632 Gammaenolase(neuralenolase)(NSE) gi|20981682 47.24 4.91 17 146 100 0.0075 1.9
635 Gammaenolase(neuralenolase)(NSE) gi|20981682 47.24 4.91 18 87 99.9 0.052 1.69
1210 Peroxiredoxin2,isoforma gi|24659879 21.88 5.66 13 201 100 0.045 (cid:1)1.29
Miscellaneous
638 DnaJhomologsubfamilyCmember7 gi|46397879 56.4 6.56 16 51 86.3 0.057 (cid:1)1.66
(tetratricopeptiderepeatprotein2)
798 Centrosomeproteincep290 gi|51890223 290.01 5.76 57 82 99.9 0.026 (cid:1)1.72
334 HypotheticalproteinLOC80129 gi|38511966 82.27 6.22 24 57 99.8 0.041 1.22
1384 Parvalbuminalpha gi|131100 12.05 4.98 9 51 86.3 0.011 (cid:1)2.21
Abbreviations:COD,cocaineoverdose;MALDI-ToF/ToF,matrix-assistedlaserdesorptionionization-timeofflight-timeofflight;NAc,nucleusaccumbens.