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 developmental time points using two-dimensional gel electrophoresis followed by mass spectrometrical protein identification 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 developmentally 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 brain2 using twodimensional 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 development3 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 twodimensional gel electrophoresis followed by mass spectrometrical analysis of kitten and cat visual cortex showed * To whom correspondence should be addressed. Prof. Dr. Gert Lubec, Medical University of Vienna, Department of Pediatrics, Wa¨hringer Gu ¨ rtel 18, A-1090 Vienna, Austria; Tel: +43-1-40400-3215 Fax: +43-1-40400-3194; E-mail:
[email protected]. † Medical University of Vienna. ‡ Core Unit of Biomedical Research. 10.1021/pr0602545 CCC: $33.50
2006 American Chemical Society
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 neuropathology. We therefore aimed to study brain protein levels at three developmental time points in a widely used and wellcharacterized rat strain with representative sample sizes. For this purpose a nonsophisticated proteomic approach, twodimensional gel electrophoresis with subsequent mass spectrometrical 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 experiments were carried out in accordance with the rules of the American Physiology Society. Animals were sacrificed by decapitation, brains were rapidly removed and complete hippocampal 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 Journal of Proteome Research 2006, 5, 3205-3212
3205
Published on Web 10/06/2006
technical notes
Postnatal Brain Protein Expression
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 1,4dithioerythritol (Merck, Germany), 1mM EDTA (ethylenediamintetraacetic 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 mm3) 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, Uppsala, 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 Spectrometry. 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 (PROTEINEER 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 octylD-glucopyranoside (OGP) and 10 mM ammonium bicarbonate) and incubated for 4 h at 30 °C. Peptide extraction was performed with 10 µL 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-hydroxy3206
Journal of Proteome Research • Vol. 5, No. 11, 2006
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 MALDITOF peptide mass fingerprint (PMF) or LIFT mode for MALDITOF/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). Database 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 methionine 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 Fisher’s 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, cytoskeleton, 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 nonsignificantly and differentially expressed proteins is provided in supplementary Table 4 (Supporting Information). The corresponding 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.
technical notes
Weitzdo1 rfer et al.
Table 1. a. Temporally Significant Expression of Proteins (Fisher’s Exact Test) accession number
P80254 Q99NA5 P39053-1 P39053-2 P39053 (total of 2) O35593 total spot volume P60901 P99026
O35737-4 P70333 Q9D6G1 P11598
3d
Metabolic proteins phosphoglycerate mutase 1 (rat) 0a/9 carbonic anhydrase II (rat) 0/9 vacuolar ATP synthase catalytic subunit A, 0/9 ubiquitous isoform (mouse) D-dopachrome tautomerase (rat) 0/9 NAD+-specific isocitrate 0/9 dehydrogenase a-subunit Cytoskeleton proteins dynamin-1 (mouse) 0/9 dynamin-1 (mouse) 0/8 dynamin-1 (mouse) 0/9
P25113-1 P27139 P50516
O35737-3
protein name
3w
3m
3d vs 3w
3d vs 3m
3w vs 3m
10/10 0/10 3/10
9/9 8/8 9/9
