Anna Bodzon-Kulakowska1
Piotr Suder1
Pawel Mak2
Anna Bierczynska-Krzysik1
Gert Lubec3
Beata Walczak4
Jolanta Kotlinska5
Jerzy Silberring1
1Neurobiochemistry Department,Faculty of Chemistry,Jagiellonian University, Krakow,Poland
2Department of AnalyticalBiochemistry, Faculty ofBiochemistry, Biophysics andBiotechnology, JagiellonianUniversity, Krakow, Poland
3Department of Pediatrics,Medical University of Vienna,Vienna, Austria
4Department of Chemometrics,Institute of Chemistry, SilesianUniversity, Katowice, Poland
5Department of Pharmacologyand Pharmacodynamics, MedicalUniversity, Lublin, Poland
Original Paper
Proteomic analysis of striatal neuronal cell culturesafter morphine administration
Using primary neuronal cell culture assays, combined with 2-D gel electrophoresisand capillary LC–MS, we identified differences in proteomes between control andmorphine-treated cells. Statistically significant differences were observed among26 proteins. Nineteen of them were up-regulated, while seven were down-regulatedin morphine-treated cell populations. The identified proteins belong to classesinvolved in energy metabolism, associated with oxidative stress, linked with proteinbiosynthesis, cytoskeletal ones, and chaperones. The detected proteins demand fur-ther detailed studies of their biological roles in morphine addiction. It is crucial toconfirm observed processes in vivo in order to reveal the nature and importance ofthe biological effect of proteome changes after morphine administration. Furtherinvestigations may lead to the discovery of new proteome-based effects of morphineon living organisms.
Keywords: Addiction / Astrocytes / Cell culture / Morphine / Neurons / Proteome /
Received: August 19, 2008; revised: December 21, 2008; accepted: December 22, 2008
DOI 10.1002/jssc.200800464
1 Introduction
Addiction to drugs such as morphine or heroin nowa-days represents a severe medical, social, and economicalproblem. It is recognized as a complex, chronic, andrelapsing mental disorder combined with changes inbehavior and chemistry of the brain [1].
Morphine is a strongly anesthetic, active substancepresent in opium. Along with heroin, which is its diace-tyl derivative converted into morphine in the brain, it ismisused because of its ability to cause euphoria andrelaxation. Those two substances are highly addictive,produce strong tolerance, physical dependence, and, as aconsequence, compulsive drug seeking and abuse. Thisstate of dependence is very difficult to cure, and attemptsto find an efficient treatment have forced scientists toinvestigate alterations caused by these drugs of addictionat various levels. One of the most promising approaches,where proper and more specific strategies of treatmentmight be found, is at the cellular level.
In contrast to the previously used analytical tech-niques, proteomics is a powerful tool for revealingchanges in protein profiles between control and drug-treated sets of samples. Proteins whose levels are affectedby addiction may serve as potential markers of this state,and thus may be used as targets for new therapies.
Proteome changes after morphine administrationhave hitherto been investigated mostly in the wholebrain [2] and its structures involved in phenomena ofaddiction and reward mechanisms, such as the prefron-tal cortex [3, 4], hippocampus, striatum [4], nucleusaccumbens [5], and in cellular fractions such as synapto-somes [6] and postsynaptic densities [7].
In our study we selected the neuronal cell culturebecause such an approach has several advantages: (i) Itensures simplification of the model, since we can observechanges in one type of cell only. Recent findings suggestthat this kind of “prefractionation” may reveal changesin protein quantities which are invisible at the level ofsuch a complex material as whole tissue homogenate [7].Therefore, there is a chance of observing low abundanceproteins and finding alterations in their levels. Largeamounts of abundant proteins and lipids typical forwhole nervous tissue are comparatively rare in suchmaterial, so its preparation for proteomics studies ismuch easier. (ii) A cellular model ensures optimal con-trol of the system during the entire experiment (concen-tration of morphine, time of action, temperature, mediacomposition, etc.). (iii) According to points (i) and (ii), the
:
Correspondence: Dr. Piotr Suder, Department of Neurobio-chemistry, Faculty of Chemistry, Jagiellonian University, Ingar-dena 3 st., 30-060 Krakow, PolandE-mail: [email protected]: +48 12 634 05 15
Abbreviations: ER, endoplasmic reticulum; HBSS, Hanks' Bal-anced Salt Solution; TCA cycle, tricarboxylic acid cycle
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
1200 A. Bodzon-Kulakowska et al. J. Sep. Sci. 2009, 32, 1200 – 1210
J. Sep. Sci. 2009, 32, 1200 –1210 Other Techniques 1201
molecular response to morphine introduction can easilybe detected.
Such a model has recently been introduced and success-fully used for studying morphine action [8]. Moreover, thecell model is commonly accepted not only for researchdevoted to morphine effects but also in many otherapproaches. It is known that the cell model may differ inits response to stimulation, as compared to a living ani-mal, therefore it should be stressed here that the obtainedresults need to be verified and application of such investi-gation cannot completely replace studies on animals orhumans. On the other hand, results may be acquired rela-tively fast and effectively with respect to cost and time,and the number of further animal tests reduced.
Morphine exerts its action on the nervous system bybinding to the l-opioid receptors. Striatum is a structureof the brain rich in this type of binding site [9]. Moreover,those receptors appear in this structure at the 14th dayof embryonic development [10], which is worth mention-ing since in our experiment the material for the cell cul-ture is obtained from the fetal rat brains at day 17. In vitrostriatal neurons form extensive networks of axons anddendrites with differentiated synapses and thus displaymany of the properties of mature nerve cells [11].
It was shown that activation of l receptors in thosecells in culture causes characteristic inhibition of adeny-late cyclase activity [12], which indicates that they couldbe a good model for studying the consequences of activa-tion of l receptors at the cellular level [13].
2 Experimental procedures
2.1 Cell culture
Rat primary neuronal cell cultures were prepared asdescribed elsewhere according to the method reported byBockaert [11] for cultures of mouse striatal neurons.Briefly, striata were dissected from 17-day-old rat embryosin Hanks' Balanced Salt Solution (HBSS; Sigma, Tauf-kirchen, Germany). The obtained pieces of tissue were col-lected and HBSS was removed by mild centrifugation.After that, striata were mechanically dissociated in amedium containing Neurobasal Medium with addition of2% v/v Supplement B-27 (Gibco), 0.5 mM L-glutamine, andan antibiotics mix (penicillin 100 IU/mL, streptomycin100 lg/mL, amphotericin 0.25 lg/mL (ICN Biomedicals,Costa Mesa, CA, USA). Dissociation was achieved initiallyby 15–20 series of pipetting the tissue fragments with aPasteur pipette with sharp edges and then with a flame-narrowed one. The cells obtained were centrifuged, super-natant was removed, and the pellet was resuspended in afresh portion of the medium. After that, the cells werecounted and plated in Layton bottles (NUNC, Germany) inamounts of about 130000 cells/cm2. Culture dishes wereprecoated overnight with poly-L-lysine (aqueous solution,
0.1 mg/mL), and on the culture preparation day they werewashed three times with water and incubated with thecell culture medium for approximately two hours beforecell plating, which improves cell adhesion. After six daysof culturing in a CO2 incubator (5% CO2, 378C, min. 95%relative humidity), when the cells formed a confluentmonolayer, half of the culture was treated with morphineat a concentration of 10 lM for five days. Then, the cellswere washed with PBS and harvested with a cell scraper(NUNC, Germany). At the end, PBS was removed by centri-fugation and each obtained cell pellet was frozen prior toanalysis. Cell culturing was repeated five times to yieldfive independent sets of samples (morphine vs. control inevery repeat). The protein content of each sample, esti-mated by the Bradford method using cell volumesobtained under the same culturing conditions, was equalto 0.9–1.0 mg/sample.
2.2 Two-dimensional gel electrophoresis
Sample preparation for 2-DE was performed as describedelsewhere [4]. Briefly, rat striatal neurons were sus-pended in 1 mL of sample buffer containing 7 M urea,2 M thiourea, 4% CHAPS, 10 mM DTT, 1 mM EDTA, 1 mMphenylmethylsulfonyl fluoride (PMSF) and a mixture ofprotease inhibitors (Roche Diagnostics, Mannheim, Ger-many), sonicated on ice (4 cycles for 0.03 min), and leftfor 1 h at room temperature in darkness to allow eachconstituent of the samples to solubilize. Then the suspen-sion was centrifuged at 140006g for 60 min at 128C toremove nucleic acids and insoluble material. Desaltingof the samples was performed using Ultrafree-4 centrifu-gal filter units (Millipore, Bedford, MA, USA). The proteincontent in the samples was measured by the Bradfordmethod.
2-DE was performed as reported by Yang [14]. The firstdimension was performed on the 18 cm immobilizednon-linear gradient strips pH 3–10. 18 cm long stripswere used (Amersham Bioscience, Uppsala, Sweden).0.7 mg of protein was applied on each one of them. Rehy-dration of strips was performed according to the manu-facturer's instructions. Focusing started at 200 V and thevoltage was gradually increased to 8000 V at 4 V/min andkept constant for a further 3 h (approximately150000 V.h in total). The strips were then equilibratedfor 15 min in a buffer containing 6 M urea, 20% glycerol,2% SDS, 2% DTT, and then for 15 min in the same buffercontaining 2.5% iodoacetamide instead of DTT. The sec-ond-dimensional separation was performed on the gra-dient (9–16%) sodium dodecyl sulfate (SDS) polyacryl-amide gels. The gels (180620061.5 mm) were run at40 mA per gel. After protein fixation in 50% methanoland 10% acetic acid for 12 h, the gels were stained withcolloidal Coomassie blue (Novex, San Diego, CA, USA) for8 h. The excess of dye was washed out with distilled
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
1202 A. Bodzon-Kulakowska et al. J. Sep. Sci. 2009, 32, 1200 – 1210
water. At the end, the gels were scanned with the ImageScanner (Amersham Bioscience). In total, five gels fromthe cell culture treated with morphine and five from thecontrol group were run.
2.3 Statistical gel image analysis and proteinquantification
PDQuest Basic version 8.0 (Bio-Rad Laboratories, Inc.) wasused for gel image analysis. In total, five gels from thecontrol group and five gels from morphine-treated cellculture were analyzed, including spot detection, match-ing, and spot quantification. All results obtained auto-matically were verified manually. Student's T-test withthe level of significance set at p a 0.05 was used to revealdifferentially expressed proteins between those twogroups. Significant spots were chosen for further analy-ses (Fig. 1). In a single case (spot number 27, Table 1),after detailed manual supervision we decided to includespot at a p value of 0.08 for further discussion.
Initially, during fully automated analysis, PDQuestsoftware identified an average of 991 spots per gel. Aftermanual verification and exclusion of the spots of poorestintensity and symmetry, PDQuest software identified theaverage number of spots per gel as equal to 430 (minimal
value 353 for a single gel, for remaining from 423 to441). Match rate between gels was better than 97%. Thecorrelation coefficient between gels was better than0.874 (for eight gels better than 0.903).
We anticipate that until common consensus concern-ing software for image analysis has been achieved, alldata should be analyzed by at least two programs. As thisis the starting point for further procedures, such strategyis crucial for the overall success of the entire experiment[15]. Following this rule, independently of the resultsobtained from the PDQuest analysis, the gels were addi-tionally analyzed by another software program recentlydeveloped by us and published elsewhere [16] as a com-plement to the PDQuest algorithms. Similarly to the pre-vious experiment involving PDQuest, the level of signifi-cance was set at p a 0.05. Detailed information concern-ing mathematical methods of analysis and the softwareare given in our previous publication [16]. Additionally,to avoid ambiguities, the results were also manually veri-fied. 2-D gel analysis is a basis for further pharmacologi-cal and biochemical research. At this stage of the investi-gations, where the search for differences between groupsis important, we considered the results obtained fromboth programs. This strategy gave more results thanfrom each software alone but, at the same time, we have
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
Figure 1. 2-D electrophoresis mastergel from morphine treated neuronsreceived after PDQuest work. Num-bers on the gel correspond to the pro-teins described in Table 1.
J. Sep. Sci. 2009, 32, 1200 –1210 Other Techniques 1203
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
Tab
le1.
Diff
eren
tially
expr
esse
dpr
otei
nsin
the
stria
taln
euro
nalc
ellc
ultu
reaf
ter
mor
phin
ead
min
istr
atio
n.
Spot
No.
Prot
ein
idA
cces
sion
No
Prot
ein
nam
ep
IM
W[D
a]M
Ssc
ore
Mat
ched
pep
tid
esSe
qu
ence
cove
rage
Prot
ein
abu
nd
ance
rati
oM
/S
Soft
war
ed
etec
tion
a)
pva
lue
Rel
ativ
ep
rote
inco
nte
nt
(M/S
)
1D
PYL5
_RA
TQ
9JH
U0
Dih
ydro
pyr
imid
inas
e-re
late
dp
rote
in5
(DR
P-5)
(ULI
P6p
ro-
tein
)6.
6062
071
934
4151
%1.
321
0.01
up
-reg
ula
ted
2D
PYL2
_RA
TP4
7942
Dih
ydro
pyr
imid
inas
e-re
late
dp
rote
in2
(DR
P-2)
5.95
6263
838
911
19%
1.20
10.
05u
p-r
egu
late
d3
DPY
L1_R
AT
Q62
950
P705
46D
ihyd
rop
yrim
idin
ase-
rela
ted
pro
tein
1(D
RP-
1)(C
olla
psi
nre
-sp
onse
med
iato
rp
rote
in1)
(CR
MP-
1)6.
6462
499
745
2037
%1.
211
0.05
up
-reg
ula
ted
4H
NR
PL_M
OU
SEQ
8R08
1O
5478
9H
eter
ogen
eou
sn
ucl
ear
rib
onu
cleo
pro
tein
L(h
nR
NP
L)6.
6560
719
107
35%
1.34
10.
03u
p-r
egu
late
d5
TCPG
_RA
TQ
6P50
2T-
com
ple
xp
rote
in1
sub
un
itga
mm
a(T
CP-
1-ga
mm
a)(C
CT-
gam
ma)
6.28
6117
970
329
34%
0.42
2/1
0.05
dow
n-r
egu
late
d
6G
BB
4_R
AT
O35
353
Q6Q
8B2
Gu
anin
en
ucl
eoti
de-
bin
din
gp
rote
insu
bu
nit
bet
a4
(Tra
nsd
u-
cin
bet
ach
ain
4)5.
7438
080
205
720
%0.
562
0.05
dow
n-r
egu
late
d
7N
DU
AA
_RA
TQ
561S
0Q
80W
E0
NA
DH
deh
ydro
gen
ase
[ub
iqu
inon
e]1
alp
ha
sub
com
ple
xsu
b-
un
it10
,mit
och
ond
rial
pre
curs
or7.
6440
753
329
1521
%0.
562/
10.
05d
own
-reg
ula
ted
8D
HE
3_R
AT
P108
60Q
66H
I8G
luta
mat
ed
ehyd
roge
nas
e1,
mit
och
ond
rial
pre
curs
or8.
0561
719
431
1626
%1.
291
0.01
up
-reg
ula
ted
9G
3P_R
AT
P047
97P0
9328
Gly
cera
ldeh
yde-
3-p
ho
sph
ate
deh
ydro
gen
ase
(EC
1.2.
1.12
)8.
1436
090
139
911
%1.
241
0.04
up
-reg
ula
ted
10G
3P_R
AT
P047
97P0
9328
Gly
cera
ldeh
yde-
3-p
ho
sph
ate
deh
ydro
gen
ase
(EC
1.2.
1.12
)8.
1436
090
130
611
%1.
351
0.05
up
-reg
ula
ted
11E
NO
A_R
AT
P047
64Q
66H
I3A
lph
a-en
olas
e(E
C4.
2.1.
11)(
2-p
hos
ph
o-D
-gly
cera
teh
ydro
-ly
ase)
(Non
-neu
rale
nol
ase)
6.16
4744
090
949
49%
1.33
10.
02u
p-r
egu
late
d
12K
PYM
_RA
TP1
1980
P119
81Py
ruva
tek
inas
eis
ozym
esM
1/M
2(E
C2.
7.1.
40)
6.63
5829
472
731
32%
1.32
10.
04u
p-r
egu
late
d13
DLD
H_R
AT
Q6P
6R2
Dih
ydro
lip
oyld
ehyd
roge
nas
e,m
itoc
hon
dri
alp
recu
rsor
(EC
1.8.
1.4)
7.96
5457
442
413
21%
1.31
10.
002
up
-reg
ula
ted
14A
LDH
2_R
AT
P118
84Q
6Q28
8A
ldeh
yde
deh
ydro
gen
ase,
mit
och
ond
rial
pre
curs
or(E
C1.
2.1.
3)6.
6356
453
696
3131
%1.
311
0.01
up
-reg
ula
ted
15D
DX
3X_M
OU
SEQ
6216
7O
0906
0A
TP-d
epen
den
tR
NA
hel
icas
eD
DX
3X(E
C3.
6.1.
–)
6.73
7345
547
114
18%
2.22
20.
05u
p-r
egu
late
d16
PDIA
3_R
AT
P115
98Pr
otei
nd
isu
lfid
e-is
omer
ase
A3
pre
curs
or(E
C5.
3.4.
1)5.
8857
044
1085
4361
%1.
221
0.03
up
-reg
ula
ted
17E
FTU
_MO
USE
Q8B
FR5
Q6P
919
Elo
nga
tion
fact
orTu
,mit
och
ond
rial
pre
curs
or7.
2349
477
831
3344
%1.
201
0.01
up
-reg
ula
ted
18R
UV
B2_
MO
USE
Q9W
TM5
Ru
vB-li
ke
2(E
C3.
6.1.
–)(
p47
pro
tein
)5.
4951
252
987
3249
%0.
442
0.05
dow
n-r
egu
late
d19
CN
DP2
_MO
USE
Q9D
1A2
Q3T
I32
Q99
PV1
Cyt
osol
icn
on-s
pec
ific
dip
epti
das
e(G
luta
mat
eca
rbox
ypep
ti-
das
e-li
ke
pro
tein
1)5.
4353
190
200
510
%0.
572
0.05
dow
n-r
egu
late
d
20TB
A1_
RA
TP6
8370
P025
51Tu
bu
lin
alp
ha-
1ch
ain
(Alp
ha-
tub
uli
n1)
4.94
5078
811
5861
52%
2.31
b)
0.01
up
-reg
ula
ted
21TB
A1_
RA
TP6
8370
P025
51Tu
bu
lin
alp
ha-
1ch
ain
(Alp
ha-
tub
uli
n1)
4.94
5078
861
320
36%
2.30
1/2b
)0.
04u
p-r
egu
late
d
22TB
B3_
RA
TQ
4QR
B4
Q8K
5B6
Tub
uli
nb
eta-
3ch
ain
(Neu
ron
-sp
ecif
iccl
ass
IIIb
eta-
tub
uli
n)
4.82
5084
242
716
19%
2.26
2/1
0.05
up
-reg
ula
ted
23TB
A2_
RA
TQ
6P9V
9Tu
bu
lin
alp
ha-
2ch
ain
(Alp
ha-
tub
uli
n2)
4.94
5080
441
459
22%
0.44
20.
05d
own
-reg
ula
ted
24A
CTB
_RA
TP6
0711
P025
70A
ctin
,cyt
opla
smic
1(B
eta-
acti
n)
5.29
4205
259
025
32%
1,66
10.
02u
p-r
egu
late
d
25A
CTZ
_MO
USE
P611
62P4
2024
Alp
ha-
cen
trac
tin
(Cen
trac
tin
)(C
entr
osom
e-as
soci
ated
acti
nh
omol
og)(
Act
in-R
PV)
6.19
4270
198
244
63%
1.20
10.
05u
p-r
egu
late
d
26ST
IP1_
RA
TO
3581
4St
ress
-ind
uce
d-p
hos
ph
op
rote
in1
(STI
1)(H
sc70
/Hsp
90-o
rgan
-iz
ing
pro
tein
)6.
4063
158
737
3236
%1.
241
0.03
up
-reg
ula
ted
27ST
IP1_
RA
TO
3581
4St
ress
-ind
uce
d-p
hos
ph
op
rote
in1
(STI
1)(H
sc70
/Hsp
90-o
rgan
-iz
ing
pro
tein
)6.
4063
158
460
1627
%1.
211
0.08
up
-reg
ula
ted
28G
RP7
8_R
AT
P067
6178
kD
agl
uco
se-r
egu
late
dp
rote
inp
recu
rsor
(GR
P78
)(H
eat
shoc
k70
kD
ap
rote
in5)
5.07
7247
318
06
7%2.
472/
10.
05u
p-r
egu
late
d
29E
NPL
_MO
USE
P081
13E
nd
opla
smin
pre
curs
or(H
eat
shoc
kp
rote
in90
kD
ab
eta
mem
ber
1)(9
4k
Da
glu
cose
-reg
ula
ted
pro
tein
)4.
7492
703
262
1312
%0.
382/
10.
05d
own
-reg
ula
ted
a)Th
eco
lum
n:“
soft
war
ed
etec
tion
”in
dic
ated
the
pro
gram
wh
ich
det
ecte
da
give
nsp
otw
ith
hig
hes
tsc
ore
(alw
ays
bet
ter
than
pa
0.05
).E
xact
valu
ear
egi
ven
inth
en
ext
colu
mn
.Th
efo
llow
ing
ann
otat
ion
was
use
d:“
1”fo
rou
row
nso
ftw
are,
“2”
PDQ
ues
tB
io-R
adso
fwar
e,ve
rsio
n8.
Wh
enb
oth
kin
ds
ofso
ftw
are
are
ind
icat
ed,t
he
one
show
ing
the
hig
her
scor
eis
firs
t,an
dth
ep
valu
eis
alw
ays
lin
ked
toth
isp
rogr
am.T
he
seco
nd
pva
lue
was
bet
wee
n0.
05–
0.1.
b)
Bot
hk
ind
sof
soft
war
ed
etec
ted
spot
wit
hp
valu
eb
ette
rth
an0.
05.
1204 A. Bodzon-Kulakowska et al. J. Sep. Sci. 2009, 32, 1200 – 1210
not overlooked data of particular importance. As themost important parameter to be revealed is a function ofa given protein and its involvement in drug dependence,the role of detected molecules will be verified by othermethods. Section 3 compares the data obtained by thetwo methods applied.
2.4 Sample preparation for mass spectrometry
Proteins excised from the gels were prepared for massspectrometric analysis as described [2]. Significant spotswere excised from the gel, transferred to the siliconizedcentrifuge tubes, and washed with water. To avoid kera-tin contamination, the procedure was performed in alaminar airflow cabinet. Since there is a relatively highdegree of reproducibility between gels, spots marked asthe same protein on “control” or “morphine” gels wereexcised from two or three gels and, depending on theircolor intensity, pooled together and processed for fur-ther analyses.
To destain the proteins, gel particles were rehydratedin 100 mM NH4HCO3for 10 min and then an equal volumeof acetonitrile was added. Tubes were vortexed for 10 minand then incubated for 10 min at 378C. During the nextstep, the liquid was removed, gel particles were shrunkwith acetonitrile and dried in a vacuum centrifuge. In-gelreduction of the proteins was achieved by incubation ofthe sample for 60 min at 608C in 10 mM DTT/100 mMNH4HCO3. The excess of liquid was removed and alkyla-tion was performed by incubation of the gel particles in55 mM iodoacetamide/100 mM NH4HCO3 for 45 min atroom temperature in the dark. After that, all liquid wasremoved, gel particles were vortexed with 100 mMNH4HCO3 for 15 min, dehydrated with acetonitrile, anddried prior to the in-gel digestion. Gel particles were re-swollen in the digestion solution (12.5 lg/mL of trypsin in50 mM NH4HCO3) for 45 min at 48C. The excess of liquidwas removed and gel particles were left overnight in50 mM NH4HCO3 at 378C. During the extraction of pep-tides, the gel particles were incubated for 15 min at 378Cin 50 mM NH4HCO3 with shaking every 5 min. After spin-ning down, the supernatant was collected and extractionwas performed twice, first with 5% formic acid/acetoni-trile (1:1 v/v) by incubation for 30 min at 308C, and the sec-ond time with a fresh amount of 5% formic acid/acetoni-trile (1:1 v/v) by ultrasonication for 30 min. The volume ofextraction solution was sufficient to cover the gel par-ticles. The total collected volume was usually between 20and 30 lL. All supernatants were pooled together andevaporated to dryness in a vacuum centrifuge.
2.5 Nano LC–MS/MS
Capillary LC–MS analysis of peptides obtained after pro-tein digestion was performed as described in [2] with
minor changes. The samples were prepared for LC–MS bydissolving them in 12 lL of 0.1% TFA solution (v/v) inwater. The LC–MS analysis was performed with the Ulti-mate/Famos/Switchos LC capillary chromatography sys-tem (LC Packings/Dionex, Amsterdam, The Netherlands).5 lL of sample was loaded onto short (0.365 mm) trapC18 column (LC Packings/Dionex, Amsterdam, The Neth-erlands) using the Famos Autosampler (LC Packings/Dio-nex, Amsterdam, The Netherlands). Loading flow rate was20 lL/min. Peptides initially purified and concentratedon the trap column were transferred to the main columnusing backflushing with solvent A (see below). Trap col-umn front-/backflushing was synchronized with theFamos/Switchos/Ultimate, an automated two-valveswitching system interfaced to the nano-LC instrumentand an autosampler (LC Packings/Dionex, Amsterdam,The Netherlands). The separations were performed on anLC Packings capillary column filled with PepMapreversed-phase material (15 cm long, 75 lm id, C18, 2–3 lm bead size, and 100 � pore size). The gradient wasformed using 98% water with 1.9% acetonitrile and 0.1%HCOOH (solvent A), and 80% acetonitrile with 19.9%water, supplemented with 0.1% HCOOH (solvent B), andwas delivered at a flow rate of 250 nL/min. The nano-HPLCsystem was controlled by Chromeleon software (Dionex).
A linear gradient was produced from 0% to 40% B in30 min, followed by 100% B from 30 to 40 min, and equi-libration of the column in 0% B from 45 min to 60 min.Total analysis time was 60 min.
The chromatographic system was coupled directly tothe Esquire 3000 quadrupole ion-trap mass spectrometer(Bruker Saxonia, Leipzig, Germany) using an in-housemanufactured “Black Dust” nanoelectrospray emitter[17]. The instrument was operated in positive-ion mode.During analysis, most intense peaks in the range of 400–1400 m/z were automatically fragmented by means ofdata-dependent fragmentation as described in the pre-vious publication [18]. Typically, about 2000 –2500 MSspectra and 400–600 MS/MS spectra were acquired dur-ing analyses; the threshold for MS/MS was set to 100 000.
2.6 MASCOT search
The spectra acquired from each Nano-LC–MS/MS wereanalyzed using Bruker's Data Analysis software. Proteinidentification was accomplished using the MASCOT ver.1.9 search engine against the Swiss-Prot and TrEMBLsequence databases. Search parameters were set as fol-lows: taxonomy: mammalian, enzyme: trypsin, with upto 1 missed cleavage, fixed modifications: carbamido-methyl, and variable one: methionine oxidation, peptidemass: monoisotopic with tolerance: 0.8 Da, and fragmentmass tolerance: 0.5 Da, possible peptide charges: 1+, 2+,and 3+, and the instrument: ESI equipped with ion trapanalyzer. Mammalia, not Rodentia or even Rattus were
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chosen in taxonomy due to the fact that protein data-bases are not complete, and there is a possibility that cer-tain protein for a rat has not been included there yet, butthere might be an equivalent for mouse or a human one.According to the high sequence homology between pro-teins of those species, there is a possibility of identifica-tion of rat protein based on its mammalian analogs.Detailed identification data are collected in SupportingInformation Table S1.
2.7 Ethical permissions
All experiments in which animals were used were pre-pared according to Polish and European CommunitiesCouncil Directives and were approved by the Local Com-mittee of Bioethics.
3 Results
We decided to use striatal neuronal cell cultures to iden-tify the changes in protein expression at a cellular levelcaused by morphine. In cultures those cells show manyfeatures of the mature nerve cells and are rich in l-opioidreceptors, which indicate that they could be a goodmodel for studying changes in proteome after morphineadministration at a cellular level. The proteins obtainedafter homogenization were separated on 2-DE gels andstained with Coomassie Brilliant Blue.
In order to identify proteins, the spots were excised,destained, in-gel reduced, alkylated, and digested withtrypsin. Peptides obtained after extraction were sepa-rated and their sequences determined by using a nano-LC–MS/MS system. Based on those data, proteins wereidentified by the MASCOT search against Swiss Prot andTrEMBL sequence databases as described in Section 2.
In total, 26 proteins were identified in 29 spots and arelisted in Table 1, with details of their MS analysis andquantification. Nineteen of them were found to be up-regulated under morphine influence; seven were down-regulated. Three proteins were represented by two dis-tinct spots with similar molecular weight but differentpI values. This suggests that they could exist as isoforms,possibly formed by post-translational modifications.
In five cases we observed two proteins in a single spot.This could be because distinct proteins have the same orvery similar physical properties (especially molecularmass and pI), and thus may co-migrate in a 2-DE gel. Judg-ing by the literature and our own observations, thisappears to be a common problem when using 2-DE. Insuch cases LC–MS/MS analysis was repeated using a sin-gle spot excised from remaining gel. Wherever necessary,we performed an additional, detailed manual analysis ofthe MS and MS/MS spectra to estimate which of the twoco-eluting proteins is crucial for the difference between
“control” and “morphine” spots. In four cases (spot num-bers 3, 4, 5, and 24) analysis was successful – it was possi-ble to determine which protein is responsible forobserved effect. In one case we were unable to distin-guish between proteins – this spot was excluded fromthe results group.
4 Discussion
Morphine exerts its influence on the cells via l-opioidreceptors. Their activation leads to initiation of, andchanges in, several signaling cascades, which at a cellu-lar level may be responsible for protein modificationsand changes in quantities, and essentially cause theaffected protein activity and function. Activation of thel-opioid receptors on exposure to morphine leads toabnormal behavior of the entire organism. The molecu-lar mechanisms underlying such dysfunction(s) are stillpoorly understood.
Morphine was shown to be able to alter protein expres-sion in several brain regions, probably through its actionon the l-opioid receptors, but these changes have nothitherto been analyzed at the level of a single cell type(i. e. in the cell culture). In our experiment we showedchanges to occur in several proteins involved in cell sig-naling, metabolic pathways, protein biosynthesis, andchaperones, as well as cytoskeletal ones. Those proteinsand their potential role in morphine addiction aredescribed and discussed. In our experiments we decidedto apply the neuronal, striatal cells in primary culture,to overcome the obstacle arising because 2-DE is able toseparate only the fraction of the total protein content. Itis estimated that about 104 proteins may be expressed ina single cell. Taking into account that 2-DE is able toreveal ca. 1500 most abundant proteins, it is obvious thatselecting a proper model for the proteomics study is veryimportant. In this case, the cell culture assures signifi-cant simplification of the sample and may allow forobservation of low abundance proteins.
This feature is clearly visible when we comparedresults obtained for beta-actin during a morphinomestudy in the brain [2], striatum [4], nucleus accumbens[5], and on the neuronal striatal cell culture level fromour study. Changes in protein quantity are undetectablein morphinome from brain and striatum, probably dueto the complexity of the samples. Limitation of the bio-logical material to the nucleus accumbens (part of thestriatum) and neuronal striatal cell culture revealedchanges in the level of this protein after morphineadministration. This observation indicates that simplifi-cation of the model may reveal changes which are unde-tectable at higher levels of cell organization.
The proteins discovered and their potential role inmorphine addiction are described below.
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4.1 Signaling proteins
Three isoforms of dihydropyrimidinase-related proteinswere found to be up-regulated in morphine-treated cellcultures: dihydropyrimidinase-related protein 2, dihydropyri-midinase-related protein 5, and dihydropyrimidinase-relatedprotein 1.
Dihydropyrimidinase-related proteins are involved in devel-opmental process of the nervous system in mammals[19]. Dihydropyrimidinase-related protein 2 plays an impor-tant role in guidance and growth of the axon, since itmediates the action of collapsin – a chemotactic factorwhich directs neuronal axons by collapsing the growthcone [20].
This protein was found to be down-regulated aftermorphine treatment in synaptosomes from cerebral cor-tex [6] and phosphorylated in morphine-dependent ratbrain frontal cortex [21] which is not in agreement withour results. According to the role of this family of pro-teins, it is possible that the influence of morphine ontheir up- or down-regulation strongly depends on thedevelopmental stage of the neurons in the nervous sys-tem.
In our experiment guanine nucleotide-binding protein subu-nit beta 4 (Transducin beta chain 4) was found to be down-regulated in neuronal cell culture exposed to morphine.Morphine acts through the l-opioid receptors and theiractivation evokes dissociation of Gi/o proteins into alphaand beta-gamma subunits. The alpha subunit is knownto inhibit several isoforms of adenyl cyclase [22], whereasthe beta-gamma dimer may also interact with adenylylcyclase, but the effect of this interaction (inhibition oractivation) depends on enzyme and gamma or beta iso-forms [23]. Guanine nucleotide-binding protein subunitbeta 1 and beta 2 were found to be down-regulated aswell in studies concerning morphinome in rat brain pre-frontal cortex during the establishment phase of mor-phine-induced conditioned place preference [3] and insynaptosomes from this part of the brain [6].
4.2 Proteins involved in energy metabolism
During our studies we found several alterations in pro-teins involved in energy metabolism. Among them wasthe one involved in oxidative phosphorylation: NADHdehydrogenase, down-regulated under morphine influ-ence, glutamate dehydrogenase up-regulated underthese conditions, and three enzymes involved in glycoly-sis: glyceraldehyde-3-phosphate dehydrogenase, alpha-enolaseand pyruvate kinase isozymes M1/M2, which levels were ele-vated after morphine administration.
NADH dehydrogenase was found to be down-regulated inrat brain prefrontal cortex during the establishmentphase of morphine-induced conditioned place preference[3] and in synaptosomes from this part of the brain [6].
Glutamate dehydrogenase was shown to be elevated dur-ing morphine administration. Our findings are consis-tent with those from studies on morphinome in synapto-somes [6], and in prefrontal cortex during the extinctionphase of the morphine-induced place preference [3].However, in the morphinome of hippocampal postsynap-tic density this enzyme was found to be down-regulated[7]. Glutamate dehydrogenase is one of the main enzymesthat takes part in neurotransmitter glutamate metabo-lism, and it is mainly thought to be the glial enzyme, butseveral studies show its presence also in the synaptic ter-minals [24]. In this compartment it is involved in energymetabolism: it oxidizes glutamate to alpha-ketogluta-rate, which is then utilized via the TCA cycle to formwater and carbon dioxide with production of ATP [25].This additional source of energy could be crucial whendown-regulation of NADH dehydrogenase was shownafter morphine administration.
Elevated levels of enzymes involved in glycolysis: glycer-aldehyde-3-phosphate dehydrogenase, alpha-enolase, and pyru-vate kinase isozymes M1/M2, might be a way of compensat-ing the high level of glucose, or may be used for acquisi-tion of the additional energy indispensable for introduc-tion of changes caused by morphine administration andto sustain these changes. There is evidence showing thatopioids may affect glucose homeostasis and usuallycause hyperglycemia [26]. It was shown that even a singleadministration of morphine is able to change the expres-sion of genes involved in glycolysis in medial striatum[27]. Changes in this group of enzymes were also shownin different papers describing the morphinome [5–7,21].
4.3 “Oxidative stress” proteins
There is a growing amount of evidence that morphineinduces oxidative stress [28] where additionally NO isinvolved. For example, this gas takes part in the acquisi-tion and expression of morphine-induced place prefer-ence [29] and in tolerance to this opioid [30]. The l3-opi-ate receptor subtype, which is activated by morphine, iscoupled to the constitutive NO synthase (cNOS) andinvolved in NO release [31]. The presence of this type ofreceptor was reported in the limbic tissues [32], i. e. it isinvolved in the reward pathways.
Glyceraldehyde-3-phosphate dehydrogenase may be involvedin alterations of glucose metabolism under morphineadministration, but, apart from playing a role in glycoly-sis, it acts as a multifunctional enzyme. Recent researchhas revealed its role in microtubule binding [33] andapoptosis [34]. Cell death is mediated in this case by S-nitrosylation of glyceraldehyde-3-phosphate dehydro-genase by NO [35].
Taking into consideration the fact that morphine is anagent known to induce apoptosis in neuronal cells [36],
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as well as our findings describing up-regulation of tubu-lin proteins during morphine administration, the pres-ence of two up-regulated isoenzymes of glyceraldehyde-3-phosphate dehydrogenase on the “morphine gels” is veryintriguing. Proteomic analysis of phosphotyrosyl pro-teins also revealed two phosphorylated isoforms of thisenzyme after morphine administration [21].
Dihydrolipoyl dehydrogenase is an enzyme mostly foundin mitochondria where it acts as a component of pyru-vate dehydrogenase, 2-oxoglutarate dehydrogenase, andglycine decarboxylase complexes [37]. However, apartfrom that role, it has NO-scavenging activity [38].Increased levels of this enzyme after morphine adminis-tration may, in spite of the above facts, indicate its rolein the process of addiction, by reducing the effect of anelevated level of NO in the cell.
Aldehyde dehydrogenase is another enzyme that could beinvolved in the response to oxidative stress, and which iselevated in the morphine-treated group. This enzymetakes part in the metabolism of aldehydes by oxidizingthem and may thus be involved in protection against oxi-dative stress [39].
4.4 Proteins involved in protein biosynthesis
Another group of proteins whose expression was foundto be changed during morphine administration wereproteins that could be linked with protein biosynthesis.This is not surprising, since we observed changes in thelevels of many different proteins. The following up-regu-lated molecules belong to this class: ATP dependent RNAhelicase DDX3X, protein disulfide isomerase A3, and elongationfactor T. Also, down-regulated proteins were found: cyto-solic non-specific dipeptidase, and RuvB-like2 (p47 protein).
Heterogeneous nuclear ribonucleoproteins bind topre-mRNA during transcription, and form ribonucleo-protein complexes essential for post-transcriptionalevents such as pre-mRNA splicing and mRNA export.They may also act as transcription factors and take partin translation [40]. There are studies suggesting a role ofheterogeneous nuclear ribonucleoprotein L in nitricoxide synthase gene expression [41] which, in connectionwith the suggested role of nitric oxide in morphineaddiction [29, 30], seems to warrant further study.
ATP dependent RNA helicases are proteins, whichunwind helical secondary structures in the RNA mole-cules. Proteins that belong to this family are involved intranscription, pre-mRNA splicing, ribosome biogenesis,mature mRNA export from the nucleus, initiation oftranslation, and RNA degradation [42].
A widely known role of protein disulfide isomerase iscatalysis of disulfide bond rearrangement in proteinsduring their biosynthesis. Due to this action, this proteincould also be considered as a chaperone and in some neu-rodegenerative disorders such as ischemia the elevated
level of this protein may play a neuroprotective role [43].An up-regulated level of this protein was also found aftermorphine administration in the whole rat brain morphi-nome study [2].
Another protein which plays a pivotal role in biosyn-thesis, and may also act as a chaperone, is elongation factorTu. Its main function during protein biosynthesis is todeliver aminoacyl-tRNA to the A-site of the ribosome. Butrecent findings indicate that this protein is mostly local-ized in mitochondria, where it interacts with misfolded,newly synthesized proteins. Under stress conditions, itmay recruit misfolded proteins to the proteasome com-plex [44]. It was shown that mutation in the EFTu geneleads to encephalopathy [45], which may suggest the neu-roprotective role of this protein.
RuvB-like 2 (p47 protein) was one of the two proteins inthis group, the level of which was lowered upon mor-phine influence. RuvB-like helicases are specific for NuA4histone acetyltransferase, a multi-subunit complex whichis responsible for histone acetylation [46]. As this modifi-cation has an established role in transcriptional activa-tion, the complex may take part in this process [47].
NuA4 complex may also play a role in DNA repair sinceRuvB, together with RuvA protein, is involved in the cor-rect processing of the Holliday Junction [48]. This struc-ture is an intermediate of homologous recombinationwhich plays an important role in repairing various typesof DNA damage [49]. The low level of RuvB-like proteinobserved in our study indicates that this process could besomehow disordered during morphine addiction.
Cytosolic non-specific dipeptidase is broadly distrib-uted in different tissues, and has wide substrate specific-ity, so it may function as a “housekeeper” in catabolismof dipeptidic substrates [50]. Its exact function remainselusive and further research is needed to reveal its role.
4.5 Cytoskeletal proteins
During our studies, in keeping with other findings, weobserved changes in the expression of cytoskeletal pro-teins. In particular, we found up-regulation of alpha-tubu-lin, and tubulin beta chains, and alpha-centractin. Anothergroup found that beta-actin was up-regulated in nucleusaccumbens after morphine administration [5]. We foundthat alpha-tubulin was up-regulated in two spots, whichmay suggest some kind of post translational modifica-tion of this protein that was previously described in theliterature. Phosphorylation of tubulin was found to be acharacteristic posttranslational modification after mor-phine administration [21] which may explain a multi-spot representation of this protein on the gels.
One spot, corresponding to alpha-2-tubulin chain, up-regulated on control gels, was somewhat surprisingbecause it was located in the area of the gel characteristicfor low molecular masses. This fact, verified by the identi-
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fied peptides covering C-terminal protein sequence only,may indicate the existence of a truncated form of thealpha-2-tubulin chain, absent in the morphine-treatedgroup.
Alpha-centractin is a subunit of the dynactin complex,which was identified as an activator of the dynein-medi-ated retrograde axonal organelle transport [51]. As it wasshown that morphine caused rapid endocytosis of l-opioid receptors in dendrites of the nucleus accumbensneurons [52], and in primary cultures of rat striatal neu-rons [53], up-regulation of this protein seems to be a nat-ural consequence of the observed processes.
Microtubules are also associated with organelle traf-ficking, so the changes in the levels of tubulins are rea-sonable too. However, in contrast to our findings, mRNAfor alpha-tubulin was showed to be down-regulated afterchronic morphine treatment in rat striatum [54]. Thisdiscrepancy suggests that more detailed studies have tobe performed to reveal the role of tubulin in a possiblecytoskeleton reorganization during morphine addiction.
4.6 Chaperones
The last identified group contains proteins which act aschaperones, or their function is associated somehowwith this molecules. Among up-regulated proteins wefound stress-induced phosphoprotein-1, which was repre-sented by two corresponding spots on the gel, and 78 kDaglucose regulated protein precursor (GRP 78). In contrast, endo-plasmin precursor (heat shock protein 90 kDa beta member 1)(GRP94) and T-complex protein 1 subunit gamma was found tobe down-regulated.
Taking into account an increase in protein biosynthe-sis, as well as a possible oxidative stress caused by mor-phine, changes in the levels of chaperones may be impor-tant from the point of view of their function in repairingof misfolded proteins.
78 kDa glucose regulated protein precursor (GRP78) and endo-plasmin precursor (heat shock protein 90 kDa beta member 1)(GRP94) are chaperones residing in the endoplasmic retic-ulum (ER); they are up-regulated after endoplasmic retic-ulum stress, and can protect the cells against cell death[55]. Endoplasmic reticulum stress is generated by anaccumulation of the misfolded proteins in the lumen ofER [56] and may lead to apoptosis. The gene for GRP78was also shown to be up-regulated during chronic mor-phine administration in the rat frontal cortex [57].
T-complex protein 1 subunit gamma is a subunit of a molec-ular chaperone (cytosolic chaperonin-containing t-com-plex polypeptide CCT) that assists in folding of about 9–15% of the newly synthesized proteins, especially thoseof cytoskeletal origin: actin and tubulin and actin-relatedproteins [58].
Stress-induced phosphoprotein 1 was found to be up-regu-lated after morphine treatment. This protein interacts
with another two chaperones: hsc70 and hsp90. Stress-induced phosphoprotein 1 is a component of a dynamic het-erocomplex chaperoning machine involved in matura-tion of different molecules [59]. This protein was also up-regulated in the hippocampus from patients with mesialtemporal lobe epilepsy, which may suggest a role of thisprotein in neurological disorders [60].
Summarizing, after detailed analyses of twenty ninespots that differ between two groups (gels), we foundtwenty six proteins belong to signaling proteins involvedin energy metabolism, associated with oxidative stress,linked with protein biosynthesis, cytoskeletal proteins,and chaperones. In three cases we found the same pro-tein in two distinct spots.
During our study we were unable to avoid some limita-tions of, e. g., 2-DE . In our experiments, we were unableto observe, e. g., changes in hydrophobic proteins, as wellas highly acidic or basic molecules [61]. Further studieswill be necessary to elucidate this specific and interest-ing branch of proteomics.
Limited resolution of 2-DE also contributed to the over-all quality of the results. Five of the significant spotsexcised from the gels contained more than one protein.In one case it was not possible to distinguish which ofthem is responsible for the observed effect so it had to beexcluded from the results pool. The solution for sur-mounting this obstacle in the future could be the use of anarrow pI gradient, thus providing better separation ofproteins with very similar physical properties, or adop-tion of another strategy involving, e. g., multidimen-sional capillary chromatography linked to mass spec-trometry.
Another aspect of the study is the software employedfor 2-D gel imaging. This problem with ambiguities aris-ing from the software used is well known among scien-tists and associated with a huge number of variables,such as: differences between gel separations, algorithmsused (e. g. warping algorithms), methods for backgroundsubtraction, spot detection, normalization, and methodsfor artifact removal. There is wide agreement in the liter-ature that the results of comparative analysis of a givenset of 2-DE gel images may be completely different,depending on the software applied or even slight differ-ences in the initial parameters such as threshold settingsor artifact detection [15, 62]. Finally, the set of dataobtained with just a single software package with lim-ited manual supervision may be extremely unreliable.The simplest way of overcoming this problem is to applyat least two independent programs and compare resultsbetween them. We undertook this strategy of using twodifferent software packages for 2-D gel comparison. Weapplied PDQuest and simultaneously our own softwarerecently developed in house and thoroughly tested [16].As expected, we found out that both programs applied inthis study are very sensitive towards initial parameters
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necessary for gel comparison. To solve this problem wedecided to perform additional manual inspection of thereceived data. Each significantly different set of spots,previously detected automatically, was manuallyinspected and finally accepted (or rejected) for proteinidentification. In Table 1 we marked spots identified byPDQuest and our own software, together with their p val-ues and information about which of them were identi-fied by both sets of software. A 23% coincidence betweenthe two kinds of software was achieved when at least oneprogram detected a spot with p value better than 0.05,and the second with a p value better than 0.1. Otherspots, detected by one kind software only, having p a
0.05, were also detected by the second program with ascore slightly higher than p = 0.1 (and vice versa).
5 Concluding remarks
Our approach to study the influence exerted by mor-phine on striatal neuronal cells in culture revealedtwenty proteins belonging to pathways not previouslylinked with the morphine action. Those proteinsdemand further detailed studies of their biological rolein morphine addiction for at least two reasons: First, allstudies which utilize the in vitro models require confir-mation of their results in vivo. Secondly, it is necessary toprove that this particular change in protein expressionrevealed during proteomics study possesses a significantbiological effect. Proteomic analysis may only indicateproteins that could be involved in particular pathway(s),but this can be regarded as no more than a hint that fur-ther, more detailed studies involving molecular biology,pharmacology, etc., are appropriate.
This work was partially supported by a grant from the Interna-tional Centre for Genetic Engineering and Biotechnology (ICGEB),Trieste, Italy (Grant CRP/POL05/02), a Vienna School of Medicinegrant, and a Jagiellonian University grant (DBN-414/CRBW/12A/2007). We also thank Ms. Maureen Cabatic for her invaluable helpduring 2-D gel electrophoresis experiments. The skilled help of Mr.Hasan Theoman is also greatly appreciated.
The authors declared no conflict of interest.
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