Changes of Hippocampal Protein Levels during Postnatal Brain

Development in the Rat

Rachel Weitzdo1 rfer,

Harald Ho1 ger,

Arnold Pollak,

and Gert Lubec*

,
Medical University of Vienna, Department of Neonatology, Vienna, Austria, and Core Unit of Biomedical
Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna, Austria
Received May 26, 2006
Abstract: Information on postnatal brain protein expression
is very limited, and we therefore compared hippocampal
protein levels in rat hippocampus at different develop-
mental time points using two-dimensional gel electro-
phoresis followed by mass spectrometrical protein iden-
tification and specific software for quantification. Proteins
from several cascades as e.g., antioxidant, metabolic,
cytoskeleton, proteasomal, and chaperone pathways were
developmentally regulated, which is relevant for design
and interpretation of protein chemical studies in the
mammalian brain.
Keywords: temporal regulation rat hippocampus postnatal
development MALDI brain development age-dependent
protein expression
Introduction
Gene expression in the brain is still holding center stage and
springing surprises. The advent of proteomics technology,
however, now challenges systematic studies at the protein
level.
1
Although there is abundant information on develop-
mentally regulated individual proteins in human and rodent
brain, a systematic study on developmental brain protein levels
during the postnatal period has not been carried out so far.
Fountoulakis and co-workers described differences in protein
levels between neonatal and adult rat brain
2
using two-
dimensional gel electrophoresis with mass spectrometrical
identification of proteins revealing a series of temporally
expressed proteins. Tsugita et al. reported spatial and temporal
expression of mouse brain proteins from the 10th week until
the 24th month of age and carried out protein profiling in rat
cerebella during development
3
using comparable technology.
4
Several proteins from different protein pathways were reported
to show temporally regulated brain protein levels. The effect
of aging on mouse pituitary protein levels were revealed by
Marzban et al.
5
by two-dimensional gel electrophoresis and
N-terminal micro-sequencing. Fluorescent difference two-
dimensional gel electrophoresis followed by mass spectro-
metrical analysis of kitten and cat visual cortex showed
differential protein levels between adult and 30 day old kittens.
6
In addition, protein profiling was carried out in a series of
different brain areas from several species, without comparison
to other stages.
7-9
However important these reports are, no high-throughput
technology was applied and results from the studies above
cannot be extrapolated to the rat. A systematic study of brain
protein levels during the postnatal period seems mandatory
as protein hallmarks of brain development would be of
importance for understanding neurobiology and neuropathol-
ogy.
We therefore aimed to study brain protein levels at three
developmental time points in a widely used and well-
characterized rat strain with representative sample sizes. For
this purpose a nonsophisticated proteomic approach, two-
dimensional gel electrophoresis with subsequent mass spec-
trometrical identification of proteins and quantification with
specific software was used. Herein we report protein profiling
and differential brain protein levels of several protein classes
in the rat thus extending and confirming knowledge on
developmental regulation of brain proteins and indeed, most
of these proteins were not shown to be temporally regulated
so far. We have been selecting the hippocampus as this is a
key area for cognitive functions and can be well-dissected in
the rat and protein expression in this area is of pivotal interest
to a broad neuroscientific forum.
Experimental Section
Animals. Three day, three week, and three month old female
Sprague-Dawley rats (Institute of Animal Breeding, University
of Vienna, Himberg, Austria) were housed in groups of up to
six per cage. Rats were maintained on 11/13 h light/dark cycle
in a temperature (21 ( 1 C) and humidity (50 ( 10%)
controlled and well-ventilated room with access to food and
drink ad libitum. The animals were bred and kept under
specific pathogen free (SPF) conditions, and all of the experi-
ments were carried out in accordance with the rules of the
American Physiology Society. Animals were sacrificed by
decapitation, brains were rapidly removed and complete hip-
pocampal tissue was taken within one minute, snap frozen,
and stored at -80 C until chemical analysis, and the freezing
chain was never interrupted.
10
The rational to select these three age groups was that these
are widely used for biochemical, genetic, and pharmacological
studies.
Sample Preparation. Hippocampal tissue samples (n ) 10
hippocampi per group, pooled left and right hippocampus from
* To whom correspondence should be addressed. Prof. Dr. Gert Lubec,
Medical University of Vienna, Department of Pediatrics, Wahringer Gurtel
18, A-1090 Vienna, Austria; Tel: +43-1-40400-3215 Fax: +43-1-40400-3194;
E-mail: gert.lubec@meduniwien.ac.at.

Medical University of Vienna.

Core Unit of Biomedical Research.

10.1021/pr0602545 CCC: $33.50 2006 American Chemical Society Journal of Proteome Research 2006, 5, 3205-3212 3205
Published on Web 10/06/2006
10 rats each that were not littermates) were homogenized and
suspended in 1.8 mL of sample buffer consisting of 8 M urea
(Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St. Louis,
MO), 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-
1-propane-sulfonate) (Sigma, St. Louis, MO), 65 mM 1,4-
dithioerythritol (Merck, Germany), 1mM EDTA (ethylenedia-
mintetraacetic acid), 1 mM PMSF, and 0.5% carrier ampholytes
Resolyte 3.5-10 (BDH Laboratory Supplies, Electran, England).
Samples were left at room temperature for 1 h and then
centrifuged at 14 000 g for 60 min, and the supernatant was
transferred into Ultrafree-4 centrifugal filter units (Millipore,
Bedford, MA) for desalting and concentrating proteins.
11,12
Protein content of the supernatant was quantified by the
Bradford protein assay system.
13
Two-Dimensional Gel Electrophoresis (2-DE). 2 DE was
performed as reported:
14
Samples of 800 g protein were applied
on immobilized pH 3-10 nonlinear gradient strips in sample
cups at their basic and acidic ends. Focusing was started at
200 V, and the voltage was gradually increased to 8000 V at 4
V/min and then kept constant for a further 3 h (approximately
150 000 Vh totally). After the first dimension, strips (18 cm) were
equilibrated for 15 min in the buffer containing 6 M urea, 20%
glycerol, 2% SDS, 2% DTT, and then for 15 min in the same
buffer containing 2.5% iodoacetamide instead of DDT. After
equilibration, strips were loaded on 9-16% gradient sodium
dodecyl sulfate polyacrylamide gels for second-dimensional
separation. The gels (180 200 1.5 mm
3
) were run at 40 mA
per gel. Immediately after the second dimension run, gels were
fixed for 12 h in 50% methanol, containing 10% acetic acid,
and the gels were stained with Colloidal Coomassie Blue
(Novex, San Diego, CA) for 12 h on a rocking shaker. Molecular
masses were determined by running standard protein markers
(Biorad Laboratories, Hercules, CA) covering the range 10-250
kDa. pI values were used as given by the supplier of the
immobilized pH gradient strips (Amersham Bioscience, Upp-
sala, Sweden). Excess of dye was washed out from the gels with
distilled water, and the gels were scanned with ImageScanner
(Amersham Bioscience).
Electronic images of the gels were recorded using Adobe
Photoshop and Microsoft Power Point Softwares.
Quantification of Protein Spots. Protein spots were outlined
(first automatically and then manually) and quantified using
the ImageMaster 2D Elite software (Amersham Biosciences,
Uppsala, Sweden). The percentage of the volume of the spots
representing a certain protein was determined in comparison
with the total proteins present in the 2-DE gel.
15
Matrix-Assisted Laser Desorption Ionization Mass Spec-
trometry. Spots were excised with a spot picker (PROTEINEER
sp, Bruker Daltonics, Germany), placed into 384-well microtiter
plates and in-gel digestion and sample preparation for MALDI
analysis were performed by an automated procedure (PRO-
TEINEER dp, Bruker Daltonics).
16
Briefly, spots were excised
and washed with 10 mM ammonium bicarbonate and 50%
acetonitrile in 10 mM ammonium bicarbonate. After washing,
gel plugs were shrunk by the addition of acetonitrile and dried
by blowing out the liquid through the pierced well bottom. The
dried gel pieces were reswollen with 40 ng/L of trypsin
(Promega, U.S.A.) in enzyme buffer (consisting of 5 mM octyl-
D-glucopyranoside (OGP) and 10 mM ammonium bicarbonate)
and incubated for 4 h at 30 C. Peptide extraction was
performed with 10 L of 1% TFA in 5 mM OGP. Extracted
peptides were directly applied onto a target (AnchorChip,
Bruker Daltonics) that was loaded with R-cyano-4-hydroxy-
cinnamic acid (Bruker Daltonics) matrix thinlayer. The mass
spectrometer used in this work was an Ultraflex TOF/TOF
(Bruker Daltonics) operated in the reflector mode for MALDI-
TOF peptide mass fingerprint (PMF) or LIFT mode for MALDI-
TOF/TOF fully automated using the FlexControl software. An
accelerating voltage of 25 kV was used for PMF. Calibration of
the instrument was performed externally with [M + H]
+
ions
of angiotensin I, angiotensin II, substance P, bombesin, and
adrenocorticotropic hormones (clip 1-17 and clip 18-39). Each
spectrum was produced by accumulating data from 200
consecutive laser shots. Those samples that were analyzed by
PMF from MALDI-TOF and were significantly different between
groups were additionally analyzed using LIFT-TOF/TOF MS/
MS from the same target. A maximum of three precursor ions
per sample were chosen for MS/MS analysis. In the TOF1 stage,
all ions were accelerated to 8 kV under conditions promoting
metastable fragmentation. After selection of jointly migrating
parent and fragment ions in a timed ion gate, ions were lifted
by 19 kV to high potential energy in the LIFT cell. After further
acceleration of the fragment ions in the second ion source, their
masses could be simultaneously analyzed in the reflector with
high sensitivity. PMF and LIFT spectra were interpreted with
the Mascot software (Matrix Science Ltd, London, UK). Data-
base searches, through Mascot, using combined PMF and MS/
MS datasets were performed via BioTools 2.2 software (Bruker).
A mass tolerance of 25 ppm and 1 missing cleavage site for
PMF and MS/MS tolerance of 0.5 Da but no missing cleavage
site for MS/MS search were allowed and oxidation of methion-
ine residues was considered. The probability score calculated
by the software was used as criterion for correct identification.
The algorithm used for determining the probability of a false
positive match with a given mass spectrum is described
elsewhere.
17
Results
A series of 190 proteins were identified in the three groups
by MALDI-TOF, and identification of statistically significant
differentially expressed proteins was verified by MALDI-TOF/
TOF.
The significantly differentially expressed proteins were from
several protein classes and pathways. Antioxidant, metabolic,
cytoskeleton, proteasome, nucleic acid binding proteins as well
as proteins from chaperones were temporally regulated.
The statistical evaluation of differences between groups was
carried out either by Fishers exact test (Table 1a), as several
proteins were undetectably low in the individual groups, or by
ANOVA followed by appropriate post-hoc tests (Table 1b).
As shown in Table 1a, proteins from metabolism, cytoskel-
eton, and the protein synthetic and handling machinery were
differentially expressed. Results are expressed as number of
present spots for an individual protein per group.
In Table 1b, means and standard deviation are given
revealing that proteins from several classes as shown above,
including antioxidant proteins, were temporally expressed.
In Table 2, information on identification of significantly
expressed proteins is listed. Results from nonsignificantly
different protein levels are shown in Table 3a,b (Supporting
Information). Information on the identification of nonsignifi-
cantly and differentially expressed proteins is provided in
supplementary Table 4 (Supporting Information). The corre-
sponding images/maps are presented in Figure 1. The stringent
conditions for considering the level of significance as P < 0.001
were selected for correcting false positives by multiple testing.
Postnatal Brain Protein Expression technical notes
3206 Journal of Proteome Research Vol. 5, No. 11, 2006
Table 1.
a. Temporally Significant Expression of Proteins (Fishers Exact Test)
accession
number protein name 3 d 3 w 3 m 3d vs 3w 3d vs 3m 3w vs 3m
Metabolic proteins
P25113-1 phosphoglycerate mutase 1 (rat) 0
a
/9 10/10 9/9 <0.0001 <0.0001 ns
P27139 carbonic anhydrase II (rat) 0/9 0/10 8/8 ns <0.0001 <0.0001
P50516 vacuolar ATP synthase catalytic subunit A,
ubiquitous isoform (mouse)
0/9 3/10 9/9 0.0007 <0.0001 ns
P80254 D-dopachrome tautomerase (rat) 0/9 10/10 8/8 <0.0001 <0.0001 0.0044
Q99NA5 NAD+-specific isocitrate
dehydrogenase a-subunit
0/9 7/7 8/8 0.0001 0.0001 ns
Cytoskeleton proteins
P39053-1 dynamin-1 (mouse) 0/9 10/10 8/8 <0.0001 <0.0001 ns
P39053-2 dynamin-1 (mouse) 0/8 10/10 9/9 <0.0001 <0.0001 ns
P39053
(total of 2)
dynamin-1 (mouse) 0/9 10/10 9/9 <0.0001 <0.0001 ns
Proteasome
O35593 -
total spot volume
26S proteasome,
non-ATPase subunit
9/9 9/9 0/8 <0.05 <0.0001 <0.0001
P60901 proteasome subunit alpha type 6 (human) 0/9 9/9 7/8 <0.0001 <0.0001 ns
P99026 proteasome subunit beta
type 4 [Precursor] (mouse)
0/9 8/8 7/7 <0.0001 <0.0001 ns
Nucleic acid binding proteins
O35737-3 heterogeneous nuclear
ribonucleoprotein H (mouse)
0/9 0/8 7/7 ns <0.0001 <0.0001
O35737-4 heterogeneous nuclear
ribonucleoprotein H (mouse)
0/9 0/9 9/9 ns <0.0001 <0.0001
P70333 heterogeneous nuclear
ribonucleo-protein H (mouse)
0/9 9/9 9/9 <0.0001 <0.0001 ns
Q9D6G1 85% identical to heterogeneous nuclear
ribonucleo-protein A/B
0/9 9/9 8/8 <0.0001 <0.0001 ns
Chaperones
P11598 protein disulfide isomerase
A3 [Precursor] (rat)
0
a
/9 7/7 0/7 <0.0001 ns <0.0001
b. Temporally Significant Expression of Proteins
3 d 3 w 3 months
accession
number protein name N mean SD N mean SD N mean SD ANOVA 3d vs 3w 3d vs 3m 3w vs 3m
Antioxidant proteins
O35244 peroxiredoxin 6 (rat) 9 0.117 0.037 10 0.397 0.076 9 0.279 0.081 <0.0001 <0.001
c
<0.05
c
ns
c
P04905 glutathione S-transferase Yb-1 9 0.040 0.042 0
e
- - 8 0.137 0.085 - 0.0002
d
-
P04906 glutathione S-transferase P (rat) 9 0.181 0.050 9 0.036 0.028 9 0.115 0.054 <0.0002 <0.001
c
ns
c
ns
c
P07632 superoxide dismutase [Cu-Zn]
(rat)
8 0.263 0.043 10 0.546 0.184 7 0.850 0.083 0.0001 ns
c
<0.001
c
ns
c
Metabolic proteins
P11980-1 pyruvate kinase, M1 isozyme 9 0.087 0.024 9 0.144 0.08 8 0.37 0.12 0.0003 ns
c
<0.001
c
<0.05
c
P11980-2
b
pyruvate kinase, M1 isozyme 9 0.29 0.084 9 0.351 0.096 8 0.958 0.493 0.0013 ns
c
<0.01
c
<0.05
c
P11980-3
b
pyruvate kinase, M1 isozyme 8 0.076 0.019 9 0.036 0.046 8 0.161 0.125 0.0206 ns
c
ns
c
<0.05
c
P11980
(total of 3)
pyruvate kinase, M1 isozyme 9 0.444 0.104 9 0.531 0.187 8 1.488 0.661 0.0004 ns
c
<0.001
c
<0.01
c
P23492 purine nucleoside phosphorylase 9 0.341 0.083 9 0.234 0.101 8 0.129 0.043 0.0007 ns
c
<0.001
c
ns
c
P25113-2 phosphoglycerate mutase 1 (rat) 9 0.245 0.069 9 0.609 0.114 8 0.722 0.108 0.0001 <0.01
c
<0.001
c
ns
c
P25113
(total of 2)
b
phosphoglycerate mutase 1 (rat) 9 0.245 0.069 9 0.733 0.14 8 0.915 0.098 <0.0001 <0.05
c
ns
c
ns
c
P31399 ATP synthase D chain,
mitochondrial (rat)
8 0.235 0.073 9 0.724 0.117 9 0.825 0.239 0.0002 <0.01
c
<0.001
c
ns
c
P48500-1 triose-phosphate isomerase (rat) 9 0.303 0.043 9 0.122 0.054 9 0.217 0.066 0.0002 <0.001
c
ns
c
ns
c
P48500-2 triose-phosphate isomerase (rat) 9 0.137 0.035 10 0.295 0.121 9 0.763 0.26 0.0001 ns
c
<0.001
c
<0.05
c
P48500-3
b
triose-phosphate isomerase (rat) 9 0.239 0.119 10 0.273 0.082 9 0.367 0.196 ns ns
c
ns
c
ns
c
P48500
(total of 3)
b
triose-phosphate isomerase (rat) 9 0.679 0.157 10 0.678 0.201 9 1.346 0.478 0.0012 ns
c
<0.01
c
<0.01
c
P97532 3-mercapto-pyruvate
sulfur-transferase (rat)
9 0.028 0.055 10 0.135 0.052 9 0.082 0.043 0.0007 0.0007
c
0.0023
c
ns
c
Cytoskeleton proteins
O89053 coronin 1A 8 0.259 0.096 10 0.558 0.115 8 0.541 0.052 0.0005 <0.001
c
<0.01
c
ns
c
P11516 lamins C and C2 (mouse) 8 0.373 0.144 10 0.076 0.038 0
e
- - 0.0003
d
- -
Q61553-1 fascin (mouse) 9 0.239 0.099 10 0.031 0.053 8 0.069 0.042 0.0003 <0.001
c
<0.05
c
ns
c
Q61553-2 fascin (mouse) 9 0.522 0.136 9 0.334 0.084 7 0.195 0.058 0.0002 ns
c
<0.001
c
ns
c
Q61553-3
b
fascin (mouse) 9 0.177 0.072 9 0.085 0.024 8 0.126 0.027 0.0048 <0.01
c
ns
c
ns
c
Q61553
(total of 3)
fascin (mouse) 9 0.938 0.184 9 0.455 0.095 7 0.395 0.053 0.0002 <0.01
c
<0.001
c
ns
c
Proteasome
Q00981 -
total spot
volume
ubiquitin carboxyl-terminal
hydrolase isozyme L1 (rat)
8 1.877 0.165 9 0.774 0.408 9 1.458 0.498 0.0006 <0.001
c
ns
c
ns
c
Q9JHW0 proteasome subunit beta
type 7 [Precursor] (rat)
9 0.293 0.063 8 0.174 0.036 9 0.126 0.035 0.0002 ns
c
<0.001
c
ns
c
Q9R1P4 proteasome subunit alpha
type 1
9 0.33 0.086 8 0.214 0.112 8 0.129 0.043 0.0016 ns
c
<0.001
c
ns
c
technical notes Weitzdo1 rfer et al.
Journal of Proteome Research Vol. 5, No. 11, 2006 3207
Discussion
As shown in the Results section, several proteins of individual
protein pathways were shown to be developmentally regulated.
Statistical analysis revealed significantly differential expression
of proteins and some structures were not even detectable in
the hippocampus of early postnatal life, as shown by Fishers
exact test. The major finding of this study is the demonstration
of proteins that have not been previously shown to be
developmentally regulated, neither in the rodent nor in the
human system. Temporal regulation of a few proteins was
Table 1. (Continued)
3 d 3 w 3 months
accession
number protein name N mean SD N mean SD N mean SD ANOVA 3d vs 3w 3d vs 3m 3w vs 3m
Nucleic acid binding proteins
O35737-1
b
heterogeneous nuclear
ribonucleoprotein H (mouse)
7 0.122 0.043 8 0.042 0.013 8 0.06 0.02 0.0017 <0.01
c
ns
c
ns
c
O3573thr7-2 heterogeneous nuclear
ribonucleoprotein H (mouse)
9 1.033 0.231 9 0.397 0.167 9 0.364 0.073 0.0002 <0.01
c
<0.001
c
ns
c
O35737
(total of 4)
heterogeneous nuclear
ribonucleoprotein H (mouse)
9 1.128 0.242 9 0.435 0.172 9 0.553 0.086 0.0001 <0.001
c
<0.01
c
ns
c
O88569-1 heterogeneous nuclear
ribonucleoproteins A2/B1
8 0.508 0.091 7 0.244 0.05 8 0.092 0.042 <0.0001 ns
c
<0.001
c
ns
c
O88569-2 heterogeneous nuclear
ribonucleoproteins A2/B1
8 0.512 0.149 7 0.161 0.03 6 0.051 0.019 0.0001 ns
c
<0.001
c
ns
c
O88569
(total of 2)
heterogeneous nuclear
ribonucleoproteins A2/B1
8 1.02 0.151 7 0.405 0.06 8 0.13 0.024 <0.0001 ns
c
<0.001
c
ns
c
P42669 transcriptional activator
protein PUR-alpha (mouse)
8 0.131 0.022 9 0.32 0.067 9 0.264 0.097 0.0003 <0.001
c
<0.05
c
ns
c
Q9CT01 heterogeneous nuclear
ribonucleoprotein D-like
8 0.377 0.045 8 0.163 0.051 8 0.148 0.039 0.0004 <0.01
c
<0.001
c
ns
c
Chaperones
P28480-1
b
T-complex protein 1,
alpha subunit (rat)
8 0.095 0.044 8 0.06 0.02 8 0.044 0.025 0.0454 ns
c
<0.05
c
ns
c
P28480-2 T-complex protein 1,
alpha subunit (rat)
8 0.872 0.31 8 0.45 0.086 8 0.174 0.058 <0.0001 ns
c
<0.001
c
<0.05
c
P28480
(total of 2)
T-complex protein 1,
alpha subunit (rat)
8 0.967 0.338 8 0.51 0.092 8 0.218 0.071 <0.0001 ns
c
<0.001
c
<0.05
c
a
Zero means undetectably low.
b
Belongs to a group of significant proteins.
c
Post-hoc test.
d
Mann Whitney-U test.
e
Technically not quantifiable.
Table 2. Identification of Significant Proteins
number protein name matches score pI (kDa)
Antioxidant proteins
O35244 peroxiredoxin 6 (rat) 25 198 5.65 24.68
P04905 glutathione S-transferase Yb-1 13 96 8.42 25.75
P04906 glutathione S-transferase P (rat) 10 149 6.89 23.43
P07632 superoxide dismutase [Cu-Zn] (rat) 7 61 5.89 15.78
Metabolic proteins
P11980 pyruvate kinase, M1 isozyme (rat) 32 474 6.69 57.68
P23492 purine nucleoside phosphorylase 16 85 5.78 32.277
P25133 phosphoglycerate mutase 1 (rat) 18 112 6.21 28.51
P27139 carbonic anhydrase II (rat) 11 182 6.88 28.98
P31399 ATP synthase D chain, mitochondrial (rat) 13 123 6.21 18.63
P48500 triosephosphate isomerase (rat) 19 255 6.51 26.78
P50516 vacuolar ATP synthase catalytic subunit A, ubiquitous isoform (mouse) 31 189 5.62 68.26
P80254 D-dopachrome tautomerase (rat) 9 81 6.15 13
P97532 3-mercaptopyruvate sulfurtransferase (rat) 11 68 5.88 32.8
Q00NA5 NAD
+
-specific isocitrate dehydrogenase a-subunit 37 190 6.46 39.61
Cytoskeleton proteins
O89053 coronin 1A 24 142 6.05 50.98
P11516 lamins C and C2 (mouse) 22 112 6.05 65.4
P39053 dynamin-1 (mouse) 32 228 7.61 97.8
Q61553 fascin (mouse) 35 269 6.21 54.4
Proteasome
O35593 26S proteasome, non-ATPase subunit 14 19 73 6.18 34.56
P60901 proteasome subunit alpha type 6 (human) 15 117 6.35 27.39
P99026 proteasome subunit beta type 4 [precursor] (mouse) 12 70 5.47 29.11
Q00981 ubiquitin carboxyl-terminal hydrolase isozyme L1 (rat) 12 84 5.12 24.77
Q9JHW0 proteasome subunit beta type 7 [precursor] (rat) 8 65 8.14 29.92
Q9R1P4 proteasome subunit alpha type 1 (mouse) 11 177 6 29.54
Nucleic acid binding proteins
O35737 heterogeneous nuclear ribonucleoprotein H (mouse) 11 381 5.89 49.19
O88569 heterogeneous nuclear ribonucleoproteins A2/B1 (mouse) 28 252 8.67 35.99
P42669 transcriptional activator protein PUR-alpha (mouse) 24 86 6.07 34.88
P70333 heterogeneous nuclear ribonucleoprotein H (mouse) 19 72 5.89 49.27
Q9CT01 heterogeneous nuclear ribonucleoprotein D-like 11 114 4.9 16.53
Q9D6G1 85% homologous to heterogeneous nuclear ribonucleoprotein A/B (acc. no: Q99020) 15 119 6.07 29.92
Chaperones
P11598 protein disulfide isomerase A3 [precursor] (rat) 27 263 5.88 56.62
P28480 T-complex protein 1, alpha subunit (rat) 39 279 5.86 60.35
Postnatal Brain Protein Expression technical notes
3208 Journal of Proteome Research Vol. 5, No. 11, 2006
Figure 1. (a-f) Master gels representing maps of antioxidant, metabolic, cytoskeleton, proteasome, nucleic acid binding, and chaperone
proteins in three months old rat hippocampus, stained with Coomassie blue are presented and developmentally regulated proteins
are highlighted in black boxes. Brains of 3 day (d), 3 week (w), and 3 month (m) old rats were used in the study.
technical notes Weitzdo1 rfer et al.
Journal of Proteome Research Vol. 5, No. 11, 2006 3209
already shown before, and these observations, obtained at the
transcriptional level or by immunochemical techniques, are
now confirmed by a proteomic approach.
Among several antioxidant proteins that showed comparable
hippocampal protein levels, glutathion-S-transferase P was
abundant in the 3 day old (3d) rat pups in contrast to
glutathione-S-transferase Yb-3. Unfortunately, results at the
mRNA steady-state level/transcripts in the rat
18
cannot be
extrapolated to the findings at the protein level due to several
technical reasons, and the whole brain rather than just the
hippocampus was used in the previous study. This problem is
inherent as most authors have been reporting systematic and
developmental regulation of proteins using the whole brain.
Moreover, the majority of studies has been focusing on fetal
brain development rather than postnatal brain development,
which, indeed, formed one rational to put the emphasis on
postnatal stages. Likewise, no systematic study on postnatal
SOD1 expression in brain has been performed, and data are
available in other organs at the enzyme activity level only.
19
Herein we reveal an increase of SOD1 in adult rat hippocampus
although the biological meaning remains elusive; these findings
are relevant when future studies at the protein level within the
period studied are designed. It may be speculated that regula-
tion of antioxidant proteins are linked to well-known and
documented metabolic changes in the brain during this period
of early life. Two more, peroxiredoxin 6 and glutathione
S-transferase Yb-1, were under developmental control.
Among a long list of metabolic proteins, only a couple of
proteins from energy, intermediary, carbohydrate, transport,
and amino acid metabolism were under age-dependent control.
Key elements of carbohydrate metabolism revealed different
levels, probably reflecting metabolic rates at the corresponding
time points. It is, however, evenly important that so-called
house-keeping genes (proteins), used for normalization of
results, vary with postnatal age and may therefore represent a
confounding factor in protein expression studies.
20
Likewise, pyruvate kinase M1 isoenzyme may reflect age-
dependent intermediary and purine nucleoside phosphorylase
age-dependent purine metabolism.
3-Mercaptopyruvate sulfurtransferase, on the other hand,
turned out to be lowest in 3d old rat pups, probably indicating
that cysteine metabolism/degradation i.e., the conversion from
cysteine in the rat pup by transsulfuration from mercaptopy-
ruvate to pyruvate, may not be a major trait in early postnatal
life.
21
This differential protein level in the hippocampus may,
however, not reflect genetically determined protein expression
but could be due to nutritional factors, as in this age group
diet is clearly distinct from 3 weeks or 3 months. This statement
may be relevant for other protein levels observed in our and
other studies.
As to intermediary metabolism, NAD+specific isocitrate
dehydrogenase was not detectable in 3d hippocampus, and this
may be interpreted as given above, and no developmental data
are available from literature searches to the best of our
knowledge.
Carbonic anhydrase II (CAII) was not detectable in 3d and
3w old rats, and the interpretation of this finding may differ
from above: CAII is the major isoenzyme in the brain partici-
pating in acid-base homeostasis, fluid transport, and myelin
synthesis.
22
Although altered fluid transport and imbalances of
acid-base homeostasis within the first days of life may be
associated with low CAII levels in 3d rats, a more plausible
explanation for 3w and 3m is the genetically determined delay
in myelination in the rodent,
23
and CAII levels are known to
be associated and strongly linked to myelination.
Mitochondrial ATP synthase, a representative component of
energy metabolism, was only significantly increasing at three
months although a trend to increase (P < 0.01) was observed.
The stringent statistical difference in the present study defining
the level of significance at P < 0.001 is the reason for simply
defining a trend of ATP synthase at P < 0.01 in the 3w
animals. The different energy requirements in different age
groups of our panels studied, nutritional equivalents, and basic
metabolic rate may be determining factors for low levels at 3d.
Bates and co-workers
24
described that in postnatal development
complexes of the electron transport chain in isolated rat brain
mitochondria was increasing with age and this finding is in
agreement with our observation. This is also in agreement with
the finding of age-controlled vacuolar ATP synthase, acting in
a different compartment.
D-Dopachrome tautomerase, cloned by Kuriyama et al.,
25
is
involved in catecholamine metabolism
26
. The gradual increase
in monoaminergic neurotransmitters
27
may be linked to D-
dopachrome tautomerase and may consequently help to
interpret the undetectably low levels at 3d.
Out of the many cytoskeleton proteins, only two fascin
expression forms were developmentally regulated. Three fascin
expression forms were observed and we did not discriminate
whether they represented post-translational modifications or
splice variants.
Two fascin spots were significantly more abundant at P <
0.001 in the group of 3d old pups. Fascin, abundant in the
rodent brain,
28
is mainly expressed in the filopodia of growth
cones and may therefore be important for architecture/
neuronal migration and early (fetal and very early postnatal)
development of the brain.
29
Coronin 1A showed the lowest
levels at 3 days, and this novel finding extends knowledge on
the morphogenetic role of coronins: coronin 3 is known to be
abundantly expressed in adult (mouse) brain.
30
Riemer and co-
workers reported developmentally regulated expression of
lamin C in drosophila.
31
Unlike in drosophila, lamin C was
disappearing to undetectably low levels at three months of
postnatal age. Dynamin-1, which is only expressed in neurons,
has been shown to be developmentally regulated,
32
and we here
add information on postnatal brain development.
The proteasome/ubiquitination system with a series of
members and individual functions plays a major role in brain
development but developmental regulation of the following
structures has not been reported as well.
Proteasomal subunit 26 S proteasome non-ATPase regulatory
subunit 14 (syn: MAD1) acts as a regulatory subunit of the 26S
proteasome functioning in ATP-dependent degradation of
ubiquitinated proteins and belongs to the M67A family of
peptidases. At the time point of 3m, it is no longer detectable
on 2DE gels, a time point when brain maturation has finished.
Proteasome subunit alpha type 1 was gradually decreasing
with postnatal age whereas proteasome subunit alpha type 6
(syn: macropain iota chain) was not detectable in the early
phase of postnatal development. It cleaves peptide bonds with
broad specificity and belongs to the peptidase T1A family.
Proteasome subunit beta type 4 (syn: macropain beta chain;
E. C. 3.4.25.1) shows similar function to the macropain iota
chain (see above), belongs to the peptidase T1B family and is
only detectable at later time points.
The proteasome subunit beta type 7 (syn: macropain zeta
chain) shows comparable structure and function but showed
Postnatal Brain Protein Expression technical notes
3210 Journal of Proteome Research Vol. 5, No. 11, 2006
highest levels in the early phase when brain maturation and
architecture are at the azimuth. Ubiquitin carboxyl-terminal
hydrolase isoenzyme L1 protein was observed with differential
regulation.
In developmental mechanisms, the concerted action of the
proteasome/ubiquitination machinery for controlled cleavage
of proteins is needed and a first preliminary clue is given by
the pattern of protein levels in the postnatal period.
The protein synthetic machinery consists of a legion of
transcription/translation factors and heterogeneous nuclear
ribonucleoproteins (hnRNPs). hnRNP H was represented by
several expression forms and four of them were under devel-
opmental control. Spot 2 showed high levels at 3d, whereas
spots three and four were undetectably low at this time point.
hn RNP H contains three RNA recognition motifs, thus
functioning in the splicing machinery by providing the sub-
strate for pre-mRNA processing events. High levels at 3d were
shown for two hn RNP A2/B1 expression forms. This structure
containing 2 RNA recognition motifs forms ribonucleosomes
along with 20 other hn RNPs and controls pre-mRNA process-
ing as well. The function of hn RNP D-like is not fully elucidated
and literature on this structure is limited; it may play a role for
spliceosome assembly.
33
High levels at 3d may indicate involve-
ment in the early stage when protein handling is the crucial
factor for brain development. Existence of this thus far hypo-
thetical protein, predicted from nucleic acid sequence, so far
has not been reported before at the protein level.
A protein 85% identical to hn RNP A/B (Q99020) has not
been described at the protein level as well and information only
proposes a role based upon the presence of a RNA binding
motif and identity to hn RNPs as a factor for RNA binding. This
probable splice variant of hn RNP A/B was not detectable at
3d and may play a role in later postdevelopmental stages.
Once proteins are synthesized, they have to be protected by
chaperones and heat shock proteins.
Protein disulfide isomerase A3 is undetectably low in the
group of 3 day old rat pups. It is catalyzing rearrangements of
disulfide bonds in proteins in the endoplasmic reticulum and
contains two thioredoxin domains. The undetectably low levels
in the 3d stage may indicate susceptibility of disulfide contain-
ing proteins to misfolding and damage during this period.
The T-complex family of proteins is very large, and only one
of the several T-complex proteins observed was developmen-
tally controlled: T-complex protein 1, alpha subunit, assists
folding of actin and tubulin and indeed, rearrangement of
cytoskeleton proteins is a major step in the formation of the
histoarchitecture in the developing brain. This molecular
chaperone showed highest levels in the 3d group, and this
finding may reflect the increased need for actin and tubulin
foldings during this phase.
Taken together, two-dimensional gel electrophoresis with
subsequent mass spectrometrical identification of proteins
represents an useful analytical tool for the search of develop-
mentally regulated proteins. Knowledge on temporal expression
of proteins is of pivotal importance as design and interpretation
of protein chemical studies relies on these facts. The technique
led to the detection of several proteins from different pathways
with age-dependent protein levels. Protein profiling/molecular
portraits of three postnatal developmental stages along with
description of so far only hypothetical structures are shown.
This first approach to concomitantly detect several time-
dependent protein levels (that may in turn represent protein
expression) in rat hippocampus can be easily extended using
larger gels or prefractionation steps. Future work may focus
on the generation of developmental milestones and hallmarks
in the individual brain regions to warrant fair designs for
protein studies and enabling correct interpretation of results.
We are currently working on temporal regulation of post-
translational modifications in the three age groups to comple-
ment information on quantitative development of protein levels
by qualitative data.
Acknowledgment. We are highly indebted to the Verein
zur Durchfuhrung der wissenschaftlichen Forschung auf dem
Gebiet der Neonatologie und Kinderintensivmedizin Unser
Kind and appreciate excellent technical assistance by Maureen
Cabatic.
Supporting Information Available: Supplementary
Tables 2-4. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Tan, S. S.; Gunnersen, J.; Job, C. Global Gene Expression Analysis
of Developing Neocortex using SAGE. Int. J. Dev. Biol. 2002, 46,
653-660.
(2) Fountoulakis, M.; Hardmaier, R.; Schuller, E.; Lubec, G. Differ-
ences in Protein Level between Neonatal and Adult Braun.
Electrophoresis 2000, 21, 673-678.
(3) Taoka, M.; Wakamiya, A.; Nakayama, H.; Isobe, T. Protein
profiling of rat cerebella during development. Electrophoresis
2000, 21, 1872-1879.
(4) Tsugita, A.; Kawakami, T.; Uchida, T.; Sakai, T.; Kamo, M.; Matsui,
T.; Watanabe, Y.; Morimasa, T.; Hosokawa, K.; Toda, T. Proteome
Analysis of Mouse Brain: Two-Dimensional Electrophoresis
Profiles of Tissue Proteins during the Course of Aging. Electro-
phoresis 2000, 21, 1853-1871.
(5) Marzban, G.; Grillari, J.; Reisinger, E.; Hemetsberger, T.; Grabherr,
R.; Katinger, H. Age-related alterations in the protein expression
profile of C57BL/6J mouse pituitaries. Exp. Gerontol. 2002, 37,
1451-1460.
(6) Van den Bergh, G.; Clerens, S.; Cnops, L.; Vandesande, F.; Arckens,
L. Flourescent two-dimensional difference gel electrophoresis and
mass spectrometry identify age-related protein expression dif-
ferences for the primary visual cortex of kitten and adult cat. J.
Neurochem. 2003, 85, 193-205.
(7) Bierczynska-Krzysik, A.; Kang, S. U.; Silberring, J.; Lubec, G. Mass
spectrometrical identification of brain proteins including highly
insoluble and transmembrane proteins. Neurochem. Int. 2006,
in press.
(8) Langen, H.; Berndt, P.; Roder, D.; Cairns, N.; Lubec, G.; Foun-
toulakis, M. Two-dimensional map of human brain proteins.
Electrophoresis 1999, 20, 907-916.
(9) Yang, J. W.; Juranville, J. F.; Hoger, H.; Fountoulakis, M.; Lubec,
G. Molecular diversity of rat brain proteins as revealed by
proteomic analysis. Mol. Divers. 2005, 9, 385-396.
(10) Lubec, B.; Kozlov, A. V.; Krapfenbauer, K.; Berger, A.; Hoeger, H.;
Herrera-Marschitz, M.; Nohl, H.; Koeck, T.; Lubec, G. Nitric oxide
and nitric oxide synthase in the early phase of perinatal asphyxia
of the rat. Neuroscience 1999, 93, 1017-1023.
(11) Fountoulakis, M.; Tsangaris, G. T.; Maris, A.; Lubec, G. The rat
brain hippocampus proteome. J. Chromatogr. B: Analy. Technol.
Biomed. Life Sci. 2005, 819, 115-129.
(12) Shin, J. H.; Gulesserian, T.; Verger, E.; Delabar, J. M.; Lubec, G.
Protein Dysregulation in Mouse Hippocampus Polytransgenic for
Chromosome 21 Structures in the Down Syndrome Critical
Region. J. Proteome Res. 2006, 5, 44-53.
(13) Bradford, M. M. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 1976, 72, 248-254
(14) Pollak, D. D.; John, J.; Schneider, A.; Hoeger, H.; Lubec, G. Strain-
dependent expression of signaling proteins in the mouse hip-
pocampus. Neuroscience 2006, 138, 149-158.
(15) Shin, J. H.; London, J.; Le Pecheur, M.; Hoger, H.; Pollak, D.;
Lubec, G. Aberrant neuronal and mitochondrial proteins in
hippocampus of transgenic mice overexpressing human Cu/Zn
superoxide dismutase 1. Free Radic. Biol. Med. 2004, 37, 643-
653.
technical notes Weitzdo1 rfer et al.
Journal of Proteome Research Vol. 5, No. 11, 2006 3211
(16) Myung, J. K.; Lubec, G. Use of Solution-IEF-Fractionation Leads
to Separation of 2673 Mouse Brain Proteins Including 255
Hydrophobic Structures. J. Proteome Res. 2006, 5, 1267-
1275.
(17) Berndt, P.; Hobohm, U.; Langen, H. Reliable automatic protein
identification from matrix-assisted laser desorption/ionization
mass spectrometric peptide fingerprints. Electrophoresis 1999, 20,
3521-3526.
(18) Abramovitz, M.; Listowsky, I. Developmental regulation of glu-
tathione S-transferases. Xenobiotica 1988, 18, 1249-1254.
(19) Schisler, N. J.; Singh, S. M. Tissue-specific developmental regula-
tion of superoxide dismutase (SOD-1 and SOD-2) activities in
genetic strains of mice. Biochem. Genet. 1985, 23, 291-308.
(20) Al-Bader, M. D.; Al-Sarraf, H. A. Housekeeping gene expression
during fetal brain development in the rat-validation by semi-
quantitative RT-PCR. Brain Res. Dev. Brain Res. 2005, 156, 38-
45.
(21) Nagahara, N.; Katayama, A. Posttranslational regulation of mer-
captopyruvate sulfurtransferase via a low redox potential cys-
teine-sulfenate in the maintenance of redox homeostasis. J. Biol.
Chem. 2005, 280, 34569-34576.
(22) Brion, L. P.; Suarez, C.; Zhang, H.; Cammer, W. Up-regulation of
carbonic anhydrase isozyme IV in CNS myelin of mice genetically
deficient in carbonic anhydrase II. J. Neurochem. 1994, 63, 360-
366. Erratum in: J. Neurochem. 1998, 70, 1777.
(23) Nogradi, A.; Jonsson, N.; Walker, R.; Caddy, K.; Carter, N.; Kelly,
C. Carbonic anhydrase II and carbonic anhydrase-related protein
in the cerebellar cortex of normal and lurcher mice. Brain Res.
Dev. Brain Res. 1997, 98, 91-101.
(24) Bates, T. E.; Almeida, A.; Heales, S. J.; Clark, J. B. Postnatal
development of the complexes of the electron transport chain
in isolated rat brain mitochondria. Dev. Neurosci. 1994, 16, 321-
327.
(25) Kuriyama, T.; Fujinaga, M.; Koda, T.; Nishihira, J. Cloning of the
mouse gene for D-dopachrome tautomerase. Biochim. Biophys.
Acta 1998, 1388, 506-512.
(26) Matsunaga, J.; Sinha, D.; Solano, F.; Santis, C.; Wistow, G.;
Hearing, V. Macrophage migration inhibitory factor (MIF)sits
role in catechilamine metabolism. Cell. Mol. Biol. (Noisy-le-grand)
1999, 45, 1035-1040.
(27) Chen, J. C.; Turiak, G.; Galler, J.; Volicer, L. Postnatal changes of
brain monoamine levels in prenatally malnourished and control
rats. Int. J. Dev. Neurosci. 1997, 15, 257-263.
(28) De Arcangelis, A.; Georges-Labouesse, E.; Adams, J. C. Expression
of fascin-1, the gene encoding the actin-bundling protein fascin-
1, during mouse embryogenesis. Gene. Expr. Patterns 2004, 4,
637-643.
(29) Sasaki, Y.; Hayashi, K.; Shirao, T.; Ishikawa, R.; Kohama, K.
Inhibition by drebrin of the actin-bundling activity of brain fascin,
a protein localized in filopodia of growth cones. J. Neurochem.
1996, 66, 980-988.
(30) Hasse, A.; Rosentreter, A.; Spoerl, Z.; Stumpf, M.; Noegel, A. A.;
Clemen, C. S. Coronin 3 and its role in murine brain morpho-
genesis. Eur. J. Neurosci. 2005, 21, 1155-1168.
(31) Riemer, D.; Stuurman, N.; Berrios, M.; Hunter, C.; Fisher, P. A.;
Weber, K. Expression of Drosophila lamin C is developmentally
regulated: analogies with vertebrate A-type lamins. J. Cell Sci.
1995, 108, 3189-3198.
(32) Cook, T.; Mesa, K.; Urrutia, R. Three dynamin-encoding genes
are differentially expressed in developing rat brain. J. Neurochem.
1996, 67, 927-931.
(33) Liu, Z.; Luyten, I.; Bottomley, M. J.; Messias, A. C.; Houngninou-
Molango, S.; Sprangers, R.; Zanier, K.; Kramer, A.; Sattler, M.
Structural basis for recognition of the intron branch site RNA by
splicing factor 1. Science. 2001, 294, 1098-1102.
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