Proteome Analysis of Primary Neurons and Astrocytes from Rat

Proteome Analysis of Primary Neurons and Astrocytes from Rat Cerebellum Jae-Won Yang,† Regina Rodrigo,‡ Vicente Felipo,‡ and Gert Lubec*,† Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria, and Laboratory of Neurobiology, Fundacioń Valenciana de Investigaciones Biome´dicas, Amadeo de Saboya 4, 46010 Valencia, Spain Received December 2, 2004

Neurons and astrocytes are predominant cell types in brain and have distinguished morphological and functional features. Although several proteomics studies were carried out on the brain, work on individual brain cells is limited. Generating individual proteomes of neurons and astrocytes, however, is mandatory to assign protein expression to cell types rather than to tissues. We aimed to provide maps of rat primary neurons and astrocytes using two-dimensional gel electrophoresis with subsequent in-gel digestion, followed by MALDI-TOF/TOF. 428 protein spots corresponding to 226 individual proteins in neurons and 406 protein spots representing 228 proteins in astrocytes were unambiguously identified. Proteome data include proteins from several cascades differentially expressed in neurons and astrocytes, and specific expressional patterns of antioxidant, signaling, chaperone, cytoskeleton, nucleic acid binding, proteasomal, and metabolic proteins are demonstrated. We herein present a reference database of primary rat primary neuron and astrocyte proteomes and provide an analytical tool for these structures. The concomitant expressional patterns of several protein classes are given and potential neuronal and astrocytic marker candidates are presented. Keywords: astrocytes • mass spectrometry • neurons • proteomics • two-dimensional gel electrophoresis

Introduction Protein profiling has been already performed as comprehensive analysis in neuroscience. In previous studies, proteome maps from human adult and fetal brain were constructed with 180 and 437 individual proteins1,2 and was carried out in human hippocampus and human frontal cortex at the tissue level in brain.3,4 In addition, several brain proteome databases have been constructed in mouse and rat.5-7 To reduce proteome complexity from brain, the most complex and heterogeneous organ, and to increase the dynamic range and specificity for proteome detection, single cell type8-10 or subcellular fractionation11,12 have been applied to extend knowledge on the brain proteome. Nonetheless, no comprehensive and systematic study has been performed to characterize brain structures and functions of different cell types in the central nervous system (CNS). Although the classic biochemical techniques were used to examine differential expression of specific proteins in different cell types in brain,13 there is limited data showing and comparing global protein expression. We therefore decided to construct two-dimensional gel electrophoresis (2-DE) maps of rat neuronal and astrocytic proteomes, and applied 2-DE and matrix-assisted laser de* To whom correspondence should be addressed. Tel: + 43-1-40 400 3215. Fax: + 43-1-40 400 3194. E-mail: [email protected]. † Department of Pediatrics, Medical University of Vienna. ‡ Laboratory of Neurobiology, Fundacioń Valenciana de Investigaciones Biome´dicas.

768

Journal of Proteome Research 2005, 4, 768-788

Published on Web 05/14/2005

sorption/ionization-time-of-flight (MALDI-TOF) and TOF/TOF with LIFT technique14 to primary cell culture systems allowing reduction of complexity to reproducibly study proteomes under defined conditions thus providing a valuable analytical method for the concomitant determination of specific protein expressional patterns, independent of antibody availability and specificity. Herein, we show specific neuronal and astrocytic expressional patterns of several protein classes and point out candidates for specific cell markers, which is of importance to identify and specify brain cells in neuroscience protocols in health and disease.

Experimental Section Primary Cultures of Rat Cerebellar Neurons. All animal procedures were approved by the Institute and met the guidelines of the European Union for care and management of experimental animals. Cultures enriched in granule neurons were obtained from cerebella of 7-day-old Wistar rats as previously described.15 Pups were killed by decapitation; cerebella were rapidly dissected in ice-cold phosphate-buffered saline (PBS) and placed in a Petri dish. After removal of PBS, tissue was cut into small fragments with a sterile surgical blade. To dissociate enzymatically, minced cerebella were incubated with 3 mg/mL Dispase, grade II (Boehringer Mannheim, Mannheim, Germany) for 30 min in a 5% CO2 incubator at 37 °C. The supernatant was removed and the cerebellar tissue was rinsed with 1 mM EDTA in PBS. Once the fragments have 10.1021/pr049774v CCC: $30.25

 2005 American Chemical Society

Proteomic Profiling of Rat Primary Neurons and Astrocytes

settled, the supernatant was removed and basal Eagle’s medium (BME) containing 40 µg/mL DNase I (Boehringer Mannheim) was added. The fragments were gently dissociated four times with a 10-mL plastic pipet and incubated for 20 min in DNase solution in a 5% CO2 incubator at 37 °C. The tissue was then dissociated again three to four times with a 10-mL plastic pipet and the cellular suspension was filtered through a mesh with a pore size of 90 µm. The filtered cell suspension was centrifuged at 400 × g for 5 min, and the cell bottom was rinsed twice with BME, by centrifuging at 400 × g for 5 min each time. Finally, the cells were resuspended in complete medium composed of BME, 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 µg/mL gentamycin, 5 µg/mL fungizone and 25 mM KCl. Cells were counted and cell viability was measured by using Trypan blue staining. Cells were seeded onto poly-L-lysine-coated plates (60-diameter) at 1.5 × 106 cells/ mL of medium and, after 20 min at 37 °C the medium containing unattached cells was removed and fresh medium was added. Proliferation of nonneuronal cells was prevented by adding 10 µM cytosine arabinoside approximately 20 h after seeding. Glucose (5.6 mM final concentration) was added to the culture medium twice a week. Postnatal cerebellar neurons prepared in this manner adhere, extend processes soon after plating and survive for long periods of time with a high viability. Cultures were kept in humidified atmosphere of 5% CO2 and 95% air at 37 °C. The cultures were used after 10-13 days in vitro (DIV). All reagents for cell culture were from GIBCO BRL (Life Technologies S. A., Barcelona, Spain). Primary Cultures of Rat Cerebellar Astrocytes. Astrocyteenriched primary cultures were prepared from cerebellum of 7-day-old Wistar rats. Briefly, dissected cerebella were finely minced and mechanically disrupted by pipetting in Dulbecco’s modified Eagle’s medium (DMEM). The cell suspension was vortexed at maximum speed for 1 min and filtered through sterile nylon mesh of 90-µm pore size to remove nondissociated tissue. Dissociated cells were resuspended in DMEM containing 10% FBS, 2 mM glutamine, 20 U/mL penicillin, 20 µg/mL streptomycin and 5 mg/mL fungizone and were seeded on polystyrene plates (60-diameter) at 20 mL/cerebellum, and were kept in humidified atmosphere of 5% CO2 and 95% air at 37 °C. Medium was changed once a week and cells were used after 10-13 DIV. The work-flows of how cell cultures are prepared is shown in Figure 1. Immunofluorescence. Neurons and astrocytes used for inmunofluorescence were seeded on coverglasses and used 12-14 days after seeding. Cells were labeled by indirect immunofluorescence as described.15 The coverglasses were rinsed with PBS at 37 °C. Cells were permeabilized with 0.4% Triton X-100 in PBS for 5 min and washed with PBS. Cells were fixed with methanol at -20 °C for 5 min. Permeabilized fixed cells were incubated with 3% BSA in PBS twice for 5 min, neurons were incubated for 2 h at 37 °C with anti-microtubuleassociated protein-2 (MAP-2; 1:1000) raised in our laboratory in rabbits and astrocytes with anti-glial fibrilary acidic protein (GFAP; 1:1000; Sigma, St. Louis, MO). Cells were rinsed three times with PBS and incubated with tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit IgG for antiMAP2 and with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG for anti-GFAP for 1 h at 37 °C. After rinsing three times with PBS for 15 min, samples were mounted on a glass slide with PBS:glycerol (2:1). Control for immunolabeling consisted of cells incubated only with secondary antibody. The fluorescence intensity in these controls was very low.

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Figure 1. Work-flows of the preparation of neurons (A) and astrocytes (B) from rat cerebellum.

Two-Dimensional Gel Electrophoresis (2-DE). Experiments were carried out in triplicate: after 3 times washing with cold PBS, harvested cells were suspended in 1 mL of sample buffer consisting of 7 M urea, 2 M thiourea, 4% CHAPS, 10 mM DTT, 1 mM EDTA, 1 mM PMSF, and a mixture of protease inhibitors (Roche Diagnostics, Mannheim, Germany). After sonication for approximately 15 s, the suspension was left at room temperature for 1 h and centrifuged at 14 000 × g for 60 min at 12 °C. Desalting was done with Ultrafree-4 centrifugal filter unit (Millipore, Bedford, MA). The protein content of the supernatant was determined by the Coomassie blue method. 2-DE was performed essentially as reported.3 Samples of 1 mg protein were applied on immobilized pI 3-10 nonlinear gradient strips (Amersham Bioscience, Uppsala, Sweden). Focusing started at 200 V and the voltage was gradually increased to 8000 V at 4 V/min and kept constant for a further 3 h (approximately 150 000 Vhr in total). The second-dimensional separation was performed on 9-16% gradient SDS polyacrylamide gels. After protein fixation for 12 h in 50% methanol and 10% acetic acid, the gels were stained with colloidal Coomassie blue (Novex, San Diego, CA) for 8 h and excess of dye was washed out from the gels with distilled water. Molecular masses were determined by running standard protein markers (Bio-Rad Laboratories, Hercules, CA) covering the range 10-250 kDa. pI values were used as given by the supplier of the immobilized pH gradient strips. MALDI-TOF and MALDI-TOF/TOFsMass Spectrometry (MS). Spots were excised with a spot picker (PROTEINEER sp, Bruker Daltonics), placed into 96-well microtiter plates and ingel digestion and sample preparation for MALDI analysis were performed by an automated procedure (PROTEINEER dp, Bruker Daltonics).14 Briefly, spots were excised and washed with 10 mM ammonium bicarbonate and 50% acetonitrile in 10 mM Journal of Proteome Research • Vol. 4, No. 3, 2005 769

research articles ammonium bicarbonate. After washing, gel plugs were shrunk by addition of acetonitrile and dried by blowing out the liquid through the pierced well bottom. The dried gel pieces were reswollen with 40 ng/µL trypsin (Promega, Madison, WI) in enzyme buffer (consisting of 5 mM octyl β-D-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-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 for MALDI-TOF peptide mass fingerprint (PMF) or LIFT mode for MALDI-TOF/TOF with a fully automated mode 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 for PMF. Those samples which were analyzed by PMF from MALDI-TOF 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. A mass tolerance of 25 ppm and MS/ MS tolerance of 0.5 Da and 1 missing cleavage site were allowed and oxidation of methionine residues was considered. The probability score calculated by the software was used as criterion for correct identification (http://www.matrixscience.com/help/scoring-help.html).

Results Characterization of Cultures by Immunofluorescence. To show purity of primary rat neurons and astrocytes, immunofluorescence analysis was performed in primary cell cultures. Typical granular cerebellar neurons were observed with FITC labeling (Figure 2A) and a polyclonal antibody against MAP-2 (Figure 2B). Over 95% of cells cultured were granule cells as identified by both their small size (8-12 µm) and rounded body shape with bipolar neurites (Figure 2, parts A and B). In typical cerebellar astrocytes labeled with FITC (Figure 2C) and antibody against astrocytic marker protein GFAP (Figure 2D), oval nuclei and a spectacular star shaped morphology were observed with fine processes radiating in all directions revealing that more than 95% of cells in the cultures were astrocytes. Protein Identification of Rat Primary Neurons and Astrocytes. Protein extracts (1 mg) of rat primary neurons and astrocytes were separated by 2-DE and several hundred spots were visualized by colloidal Coomassie blue staining. After ingel digestion, 428 protein spots corresponding to 226 different proteins in neurons and 406 protein spots (228 individual proteins) in astrocytes were unambiguously identified using MALDI-TOF and TOF/TOF with Mascot database searching and 770

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Figure 2. Morphology of cerebellar neurons and astrocytes in culture. A, B: Typical granular cerebellar neurons labeled with FITC (fluorescein isothiocyanate, green) (A) and antibody against MAP-2 (red) (B), showing extensive ramification of the dendritic system. C, D: Typical cerebellar astrocytes labeled with FITC (fluorescein isothiocyanate, green; (C) and GFAP immunoreactivity (D), showing oval nuclei, and a spectacular star shaped morphology with fine processes radiating in all directions.

assigned to the 2-DE gel images (Figure 3). Data obtained from MS and MS/MS analysis are presented in Table 1 including matched peptide numbers (matches), theoretical and observed pI and molecular weight (MW) values, number of identified spots and statistically significant Mascot scores (P < 0.05). Classification of Identified Proteins and Overlapped Proteins. Identified proteins in neurons and astrocytes were categorized into several functional groups based on their representative biological roles (Table 1, Figure 4). The identified metabolic proteins were included in important pathways such as citric acid cycle, glycosis, glycogenesis, mitochondrial respiratory chain, pentose phosphate pathway, and fatty acid β-oxidation cycle. Table 2 shows 7 superimposed spots (4 proteins were from neurons and 3 from astrocytes) and two different proteins from the same spot were identified by MS and MS/MS analysis with subsequent Mascot search. Deduced peptide sequences from two significantly identified proteins from the same spot were submitted to BLAST searches and aligned (www.ncbi.nlm. nih.gov/BLAST; http://clustalw.genome.jp/) to find identity to known proteins. No significant identity between two proteins from the same spot was detected. Identification of Cell Specific Proteins. Out of 335 proteins that were identified from neurons and astrocytes, 119 proteins were expressed in both cells and 107 proteins and 108 were only identified in neurons and astrocytes, respectively (Table 1). After database and literature (PubMed) searches we found some cell-specific candidate marker proteins. Neuronal tropomodulin (P70566), kinesin heavy chain isoform 5C (P28738), neuron-specific class III β-tubulin (Q8K5B6), ubiquitin carboxyl-terminal hydrolase isozyme L1 (PGP9.5; Q9R0P9) were identified in neurons, and glial fibrillary acidic protein (GFAP; P47819) and eukaryotic translation initiation factor 1 (eIF1; P48024) were observed in astrocytes. Eukaryotic translation initiation factor 1 was unambiguously identified using Mascot database. The database search was performed with 5 matching tryptic peptides (Figure 5A) which covered 47% of the total eIF1


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Figure 3. 2-DE maps of rat primary neurons (A) and astrocytes (B). The identified proteins are designated by their SWISS-PROT or NCBI accession number. The names of the proteins are listed in Table 1. Superimposed spots representing two distinct proteins are indicated by a circle. (Table 2). Journal of Proteome Research • Vol. 4, No. 3, 2005 771

772

Journal of Proteome Research • Vol. 4, No. 3, 2005 protein name (synonym)

P28480 P80314 P80316 P80318 P63039

TCPA_RAT TCPB_MOUSE TCPE_MOUSE TCPG_MOUSE CH60_RAT

T-complex protein 1, R subunit (TCP-1-R) T-complex protein 1, β subunit (TCP-1-β) T-complex protein 1, subunit (TCP-1-) T-complex protein 1, γ subunit (TCP-1-γ) 60 kDa heat shock protein, mitochondrial [Precursor] (Hsp60) (60 kDa chaperonin) (Heat shock protein 60) Q9CQN1 TRAL_MOUSE Heat shock protein 75 kDa, mitochondrial [Precursor] (HSP 75) (TRAP-1) (TNFRassociated protein 1) P48721 GR75_RAT Stress-70 protein, mitochondrial [Precursor] (75 kDa glucose regulated protein) (GRP 75) (Mortalin) P06761 GR78_RAT 78 kDa glucose-regulated protein [precursor] (GRP 78) (Immunoglobulin heavy chain binding protein) (BiP) P08113 ENPL_MOUSE Endoplasmin precursor (Endoplasmic reticulum protein 99) (94 kDa glucose-regulated protein) (GRP94) P10111 PPIA_RAT Peptidyl-prolyl cis-trans isomerase A (EC 5.2.1.8) (PPIase) (Rotamase) (Cyclophilin A) (P31) P11598 PDA3_RAT Protein disulfide isomerase A3 [Precursor] (EC 5.3.4.1) (Disulfide isomerase ER-60) (ERp60) (ERp57) (HIP-70) Q9DC41 Q9DC41 Heat shock 70 kD protein 5 (Glucose-regulated protein, 78 kD) P08109 HS7C_MOUSE Heat shock cognate 71 kDa protein

O88376 SODC_RAT Q9Z0V6

SWISS-PROT entry name

Type II peroxiredoxin 1 Superoxide dismutase [Cu-Zn] (EC 1.15.1.1) PRx III [95% homologous to P20108, peroxiredoxin 3] Q9Z0V5 Q9Z0V5 PRx IV [93% homologous to O08807, peroxiredoxin 4] Q9R063 PDX5_RAT Peroxiredoxin 5, mitochondrial [Precursor] (Prx-V) O35244 PDX6_RAT Peroxiredoxin 6 (Antioxidant protein 2) P11232 THIO_RAT Thioredoxin P04906 GTP_RAT Glutathione S-transferase P (EC 2.5.1.18) (GST 7-7) (Chain 7) (GST class-pi) Q91W90 TXN5_MOUSE Thioredoxin domain containing protein 5 [Precursor] Q9CQM9 TXL2_MOUSE Thioredoxin-like protein 2 (PKC- interacting cousin of thioredoxin) (PKC-θ-interacting protein) Q63716 PDX1_RAT Peroxiredoxin 1 (Thioredoxin peroxidase 2) P07895 SODM_RAT Superoxide dismutase [Mn], mitochondrial [Precursor]

O88376 P07632 Q9Z0V6

no.

no. of matches scorec spot

15.4 23.5 11.0

8.94 22178.69 7.45

5.40 6.02 5.97

pI

73.8 38.8 65.0

5.97 73859.67 5.53-5.62 5.07 72346.99 4.76 5.37

15.2 55.0 76.0 98.0 73.0 70.0 26.9 36.3

8.37 17743.13 6.26, 6.70 5.88 56623.37 5.70-5.94 5.01 72423.00 5.13 5.37 70871.07 5.48 5.45 5.44, 5.48 7.85 6.35

4.74 92475.77 4.85 4.75 4.68 106.0 54.2 35.7

73.0

60.0 59.0 50.5 58.2 63.0 49.0 58.2-55.5

6.25 80208.96 6.07, 6.12

Chaperone 5.86 5.96 6.12, 6.40 5.59, 5.64 6.28 5.94 5.48-5.56

30

28

28

9

17

16

31

20

28 22 22 25 22

2 4

164e 246e

6

3

78e

279e

2

155e

1

3

434e

185

2

2 2 2 1 4 109

267e 208e 169e 108 328e

15.1 15.3

50.0-108.0

29.7 79.0

73.0

78.8

61.0 51.6 31.0 68.5 59.0 55.0 45.6

21.3 21.1

40.4

5.92 6.16 5.75-5.90 29.3 31.2 56.0

6.63 7.70-8.60

4.53-5.76

6.13 5.14, 5.19

5.57

6.00

5.92 6.06 6.10 6.20 5.52 5.70 5.91

8.53 7.90

8.27 22109.41 8.96 24674.05 60359.65 57477.24 59624.08 60629.98 60955.49

5.57

5.42 37778.38

5.86 5.97 5.72 6.28 5.91

5.52

5.51 46415.48

44.0

23.0 22.7

15.4

5.89 7.40

1 1

7.12

25.2

24.1

94 79

1

166e

5.96

20.8 15.1 23.2

5.77, 5.83

10 10

1

2 1 1

146

265e 306e 130e

MW (kDa)

5.65 24687.40 5.78 4.80 11542.28 4.75 7.30 23307.70

9

15

24.9

MW (kDa)

6.18 31007.48 5.99

pI

observedb

observedb

13 14 11

MW (Da)

astrocytes

neurons

Antioxidant protein 5.20 21791.67 5.32 21.0, 20.1 5.89 15780.47 5.90 15.1 7.14 28321.43 6.00 22.5

pI

theoreticala

Table 1. List of Proteins Identified from Rat Primary Neurons and Astrocytes

26

9

19

18

24

23

26 23 23 12 27

17 11

15

11

13

23

14

15

12 10 14

2

230e

299e

87e

104

179e

280e

126

173 272e 132e 75 208e

190 146e

105

5

4

4

3

1

1

1 1 1 1 3

1 1

1

1

2

214e

76

1

1

144e 297e

1 1 1

309e 94 135e

no. of matches scorec spot

research articles Yang et al.

O35814

HS72_RAT

HSB1_RAT

TCPZ_MOUSE PDI_RAT

PDA6_RAT

CRAB_RAT

TBA1_MOUSE

AAA37742 O35814

P14659

P42930

P80317 P04785

Q63081

P23928

P02551

ACTB_MOUSE

PFD2_MOUSE O88600

O70591 O88600

P60710

Q8CGW7 Q6TUG0

Q8CGW7 Q6TUG0

Q13885

Q9WUD1

Q9WUD1

Q13885

Osmotic stress protein 94 (Heat shock 70-related protein APG-1) Carboxy terminus of Hsp70-interacting protein (STIP1 homology and U-Box containing protein 1) Immunophilin XAP2 LRRGT00084 [99.44% identity to Q99KV1, DnaJ homologue subfamily B member 11 precursor] Prefoldin subunit 2 Ischemia responsive 94 kDa protein

OS94_MOUSE

P48722

Actin, cytoplasmic 1 (β-actin)

β tubulin (Tubulin, β polypeptide) (Novel protein similar to tubulin β polypeptide (TUBB), variant 1)

5.29

4.78

Tubulin R-1 chain

41736.73

49906.97

20088.83

Cytoskeleton 4.94 50135.63

47220.18

58004.26 56951.34

22892.67

69528.50

18943.09 62570.10

16533.71 94056.53

37666.95 40494.99

34909.31

6.76

4.95

6.63 4.82

6.12

5.44

4.66 6.40

6.20 5.13

6.08 5.92

5.71

94382.07

70928.12

84814.91

Protein disulfide isomerase A6 [Precursor] [Fragment] (Calcium-binding protein 1) (CaBP1) R Crystallin B chain (R(B)-crystallin)

Glucose-regulated protein 78 P60 protein (Stress-induced-phosphoprotein 1) (Hsp70/Hsp90-organizing protein) Heat shock-related 70 kDa protein 2 (Heat shock protein 70.2) (Testis-specific heat shock protein-related) (HST) Heat-shock protein β-1 (HspB1) (Heat shock 27 kDa protein) (HSP 27) T-complex protein 1, ζ subunit (TCP-1-ζ) Protein disulfide-isomerase [Precursor] (EC 5.3.4.1) (PDI) (Prolyl 4-hydroxylase β subunit)

5.43

dnaK-type molecular chaperone hsp72-ps1 - rat

S31716d 5.54

4.93

Q91XW0

Heat shock protein 86

Q91XW0

5.13 5.22 5.40 5.42 6.75 4.38-5.60 5.87-5.97 5.88, 6.00 4.85-4.66 5.10-5.43 5.75 5.90 5.74, 5.96 5.43-5.57 4.80 6.18 4.98-5.35 5.43, 5.46 5.44

6.00 5.40 4.99 4.87

6.20 6.16

5.76

5.48, 5.52 5.73 5.00 4.70 5.62, 5.67

15.0 15.3 14.1 25.5 52.0 49.0-55.0 44.0-47.2 36.5 15.2-10.5 51.0 38.8, 41.0 42.0 36.0 32.3-34.0 21.5, 25.0 43.2 28.8-34.3 62.0 28.8

15.9 120.0 80.0 66.7

34.3 39.0

32.5

89.0 42.8 41.1 32.4 107.0

17

36

30

4 25

11 11

19

28

12

31

21

3

414e

160e

24

1 3

43e 137e

298e

1 1

63 60

1

2

166e 85

2

3

181e

172

5.41-5.66 5.47 5.46 5.48 5.60 5.43 4.63-5.86

5.50-5.74 6.60 4.91 5.02 5.52

4.89 4.96 5.57-5.95 5.35 5.60

6.81

6.67 5.00 4.97 5.28 6.19

5.79

6.73

4.60 6.26, 6.50

23.6-19.8 36.9, 120.0 31.0 58.0 54.0 49.5 36.9-48.0

34.3-42.0 18.2 14.6 25.0 52.3

15.9 16.1 46.2-59.1 57.1 36.4

18.7

62.0 10.5 57.0 25.0 41.0

25.2

8.50

20.0 65.0

17

31

21

7

24

18 13

13

8

10 32

1 1 3 1 1

190e 129e 79 170e 85e

160e

228e

19

8

9

1

161e

301e

1 2

75 288e


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Journal of Proteome Research • Vol. 4, No. 3, 2005 773

774 protein name (synonym)

Q61274

ACTZ_HUMAN

CAZ2_MOUSE CAPB_MOUSE COF1_RAT O35303 Q8VHK3 Q80V75 H2B2_MOUSE

PRO2_RAT VIME_RAT

WDR1_MOUSE Q9JMJ4

TMO2_RAT

Q8K5B6

TBA6_MOUSE

Q9D9Y2 TBB3_MOUSE

TBB4_MOUSE

TBB5_HUMAN

CLCB_RAT COF2_MOUSE DREB_RAT DYN1_RAT

P61163

P47754 P47757 P45592 O35303 Q8VHK3 Q80V75 P10854

Q9EPC6 P31000

O88342 Q9JMJ4

P70566

Q8K5B6

P05216

Q9D9Y2 Q9ERD7

Q9D6F9

P05218

P08082 P45591 Q07266 P21575

MW (Da)

pI


Clathrin light chain B (Lcb) Cofilin, muscle isoform(Cofilin 2) Drebrin (Developmentally regulated brain protein) Dynamin-1 (EC 3.6.5.5) (D100) (Dynamin, brain)

Tubulin β-5 chain

Tubulin β-4 chain

Tubulin R-7 Tubulin β-3

WD-repeat protein 1 (Actin interacting protein 1) Rattus norvegicus neuronal differentiationrelated [99.80% identity to Q99KP6, Nuclear matrix protein SNEV] Neuronal tropomodulin (N-Tmod) (Tropomodulin 2) Tubulin β-4 chain (Neuron-specific class III β-tubulin) Tubulin R-6 chain (R-tubulin 6)

Profilin II Vimentin

35.9

40.0

65.0 52.5

33.6 29.0 16.1 79.0 79.0 49.4 14.2 14.8 12.5 41.8-43.0

43.0

13.9

42.0 42.0 40.0 27.8 25.3

8 11 23 28

7

24

11 32

15

26

17

20 15

9 21

18 16 12 28 19 16 11

19

7

26

matches

scorec

no. of spot pI

3 4

2 6

1 1 1 4

166e 273e

165e 73e

75e 164e 274e 180e

2

1

207e 80

1

158e

1 3

147e 223e

1 1

1 1 1 1 1 1 2

250e 110 104 119 129e 84 63

221 130

1

1

8

283e

65

264e

5.82 4.90 5.00-5.30 6.17-6.34 6.14

5.60 5.63 5.79-8.28 5.74 5.97 6.37, 6.53 5.71

5.53 5.31-5.48 49.5 5.47-5.53 5.45 5.74 5.76 6.19 5.25 5.35 6.26

12.8 31.0-45.0 46.8-57.0 68.0 55.2

33.6 29.9 16.4 48.0 81.5 51.0 14.5

31.0, 36.6 42.1-49.0 4.50 18.8-28.4 57.2-69.0 43.5 44.0 43.3 14.8 14.3 44.5

MW (kDa)

16 13

15 30

10 10 9 15 20 25 9

15

7

23

matches

observedb

MW (kDa)

astrocytes

neurons observedb

4.96 49909.36 5.89 37.8 6.32 54.0 5.76 44049.26 5.66-5.60 22.0-17.2 4.82 50418.65 5.30 30.0 5.31 28.8 5.55 33.0 5.84 37.5 4.78 49551.75 5.44 33.2 5.61 36.2 4.78 49670.82 4.58 12.7 4.35 51.5 4.60 51.0 6.04 36.0 5.52, 5.84 52.0 4.56 25117.44 4.20 32.0 7.66 18709.59 7.55 16.3 4.46 77471.96 4.25 130.0 6.32 95927.48 6.03-6.51 99.0

4.88 50303.05 5.82

5.34 39491.83 5.52

6.11 66406.70 6.40 6.17 55247.89 6.21

5.63 5.62 6.17 6.45 6.03 6.51 4.09 6.72 6.78 14870.93 5.80 5.06 53601.54 4.72-4.90

32835.87 31214.28 18532.52 83908.37 54173.91 51405.46 13804.97

6.19 42613.74 6.35

R-centractin (Centractin) (Centrosome-associated actin homolog) (Actin-RPV) (ARP1) F-actin capping protein R-2 subunit (CapZ R-2) F-actin capping protein β subunit (CapZ β) Cofilin, nonmuscle isoform Dynamin-like protein Ezrin [Fragment] Fscn1 protein [Fragment] [Fascin] Histone H2B 291B 5.58 5.47 8.22 6.64 6.16 6.57 10.32

5.30 16769.06 4.89

Cytoskeleton 5.31 41792.84 3.80 5.36, 5.43 5.72, 5.84 5.45, 5.53 5.49

pI

theoreticala

R-cardiac actin [Fragment]

ACTG_HUMAN Actin, cytoplasmic 2 (γ-actin)


Q61274

P02571

no.

Table 1 (Continued)

1 8 3 1

191e 74

1 1 3 1 1 2 1

1

2

22

no. of spot

125 424e

91 75 82e 69 206e 288e 74

133e

80e

257e

scorec


KF5C_MOUSE

LAM1_RAT

Q99N28

SPCN_MOUSE Q91ZZ3 COAC_MOUSE ARP3_MOUSE AAC1_RAT CLP2_MOUSE Q99LB4 Q7M0E3 FLNA_MOUSE

Q7TN32 GFAP_RAT

LAMA_MOUSE

LAMC_MOUSE Q99MZ8 PDL1_RAT

PRO1_MOUSE

P70524 Q922F4 SLI2_MOUSE Q91VU2 TPM4_RAT TBA2_MOUSE Q64727

AKA1_RAT

DHAM_RAT

O88989 DLDH_MOUSE

G6PD_RAT Q9QWU4

IDHC_RAT

IVD_RAT

LDHB_RAT

Q99NA5

P28738

P70615

Q99N28 C34787 P16546 Q91ZZ3 Q9CQI6 Q99JY9 Q9Z1P2 Q08093 Q99LB4 Q7M0E3 Q8BTM8

Q7TN32 P47819

P48678

P11516 Q99MZ8 P52944

P10924

P70524 Q922F4 Q9R059 Q91VU2 P09495 P05213 Q64727

P51635

P11884

O88989 O08749

P05370 Q9QWU4

P41562

P12007

P42123

Q99NA5

Alcohol dehydrogenase [NADP+] (EC 1.1.1.2) (Aldehyde reductase) (3-DG-reducing enzyme) Aldehyde dehydrogenase, mitochondrial [Precursor] (EC 1.2.1.3) (ALDH class 2) (ALDH1) (ALDH-E2) Cytosolic malate dehydrogenase (EC 1.1.1.37) Dihydrolipoyl dehydrogenase, mitochondrial [Precursor] (EC 1.8.1.4) (Dihydrolipoamide dehydrogenase) Glucose-6-phosphate 1-dehydrogenase (G6PD) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Isocitrate dehydrogenase [NADP] cytoplasmic (EC 1.1.1.42) (Oxalosuccinate decarboxylase) (IDH) (IDP) Isovaleryl-CoA dehydrogenase, mitochondrial [precursor] L-lactate dehydrogenase B chain (EC 1.1.1.27) (LDH-B) (LDH heart subunit) (LDH-H) NAD+-specific isocitrate dehydrogenase a-subunit

Rat smooth muscle R-tropomyosin 97.98% identity to P09653, Tubulin β-5 chain Skeletal muscle LIM-protein 2 (SLIM 2) (FHL-3) Transgelin 2 (SM22β) Tropomyosin R 4 chain (Tropomyosin 4) (TM-4) Tubulin R-2 chain (R-tubulin 2) Vinculin (Metavinculin)

Lamins C and C2 LASP-1 PDZ and LIM domain protein 1 (LIM domain protein CLP-36) (C-terminal LIM domain protein 1) (Elfin) Profilin I

Lamin A

Nectin-like protein 1 (Membrane glycoprotein) tropomyosin 3 R, brain - rat Spectrin R chain, brain [Fragment] (R-II spectrin) β-synuclein (Synuclein, β) Coactosin-like protein Actin-like protein 3 (Actin-related protein 3) R-actinin 1 (Nonmuscle R-actinin 1) Calponin H2, smooth muscle Capg protein Destrin Filamin A (R-filamin) (Filamin 1) (Endothelial actin-binding protein) (ABP-280) (Nonmuscle filamin) Fscn1 protein Glial fibrillary acidic protein, astrocyte (GFAP)

Kinesin heavy chain isoform 5C (Kinesin heavy chain neuron-specific 2) Lamin B1

14826.02

65446.08 29970.40 35525.17

74209.51

54507.98 49943.07

42964.31 28343.45 167553.20 14051.61 15943.91 47357.15 102960.33 33155.51 38768.88 18387.39 281193.50

66475.25

109240.52 5.36 5.49 5.96 4.52 5.20 4.21 5.06

6.00

6.46

5.70

8.03

5.58

5.97 8.14

6.16 7.97

6.63

39613.65

36481.18

46435.46

33174.03

59244.52 35827.99

36483.11 54212.32

56488.42

5.66 5.81

5.69

6.22

6.55, 6.80

6.07 6.35-8.92

6.04, 6.50 7.00

5.79 6.08

4.90 32138.99 4.80 50090.37 5.75 31817.56 8.40 22395.43 4.66 28509.70 4.94 50165.66 5.77 116586.17 Metabolic proteins 6.81 36374.69 6.90, 7.25

8.50

6.37 6.61 6.56

6.54

6.44 5.35

5.50 4.74 5.27 4.38 5.28 5.61 5.23 7.53 6.47 7.78 5.68

5.16

5.84

37.0 36.8

34.6

40.0

42.0

56.8 32.3-34.8

33.6 52.0

50.0 49.5

36.0

67.2 56.2 26.0 29.0 42.0 16.6 14.0

130

18

41

15

22

35 21

12 15

23

17

6 11 16 6 12

23

27

2 1 1 9

186e 118e 366e 198e

1 1 2

82 461e 260e

2

2

175e

131

2

1 1 1 1 1

65e 100e 78 203e 152e

158e

2

1

119

118

5.79

5.69

6.15

6.40, 6.60

5.94-6.01 6.54-8.30

6.00, 6.05 6.68

5.88-6.02

6.80

7.60 8.28 4.70 5.63 5.65 8.63 4.75 5.63 5.78-5.98

6.20 4.70-5.40 5.51 5.49 6.40 6.59 6.30, 6.55 6.50 6.53

5.67 5.69 7.75 6.12 6.41 6.19 6.05

37.9

35.9

41.0

43.0

58.9 33.0-35.5

34.2 55.5

50.5-52.5

37.0

12.6 12.9 34.0 34.2 35.7 20.1 30.8 18.5 73.0-118.0

50.8 25.8-15.7 38.6 49.6 62.0, 73.0 72.9 61.0 36.1 34.7

48.5 109.0 33.0 41.0 16.0 24.6 70.0

19

15

15

23

29 23

10 14

29

15

9 25 12 17 19 10 33

13

18 15 15

15

29 22

12 24 23 10 14 11

1 1 1 1 1 1 5

79e

254e

77

1

1

1

2

256e 93

2 8

2 1

2

265e 268e

66e 95e

388e

190e

1

2

135e 248e 100e 109 222e 163e 228e

2 1 1

3

1 6

1 1 1 1 1 2

105 87 133

72

244 206e

75 123 120 76 105 113e


research articles


776


KPY1_RAT KPY2_RAT

KCY_MOUSE

PGK2_RAT

PMG1_RAT

APT_RAT

HPRT_RAT

THTM_RAT Q91Y38 OAT_RAT

Q8K2S8

AR72_RAT

PA1B_RAT

TPIS_RAT

IDI1_RAT ALFC_RAT

P11980 P11981

Q9DBP5

P16617

P25113

P36972

P27605

P97532 Q91Y38 P04182

Q8K2S8

Q8CG45

O35264

P48500

O35760 P09117

Isopentenyl-diphosphate δ-isomerase 1 Fructose-bisphosphate aldolase C (Brain-type aldolase)

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) (HGPRT) (HGPRTase) 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) UDP-N-acetylglucosaminyltransferase Ornithine aminotransferase, mitochondrial [Precursor] (EC 2.6.1.13) (Ornithinesoxo-acid aminotransferase) Similar to ubiquinol-cytochrome c reductase core protein 1 Aflatoxin B1 aldehyde reductase member 2 (rAFAR2) Platelet-activating factor acetylhydrolase IB β subunit (PAFAH β subunit) (PAF-AH R 2) Triosephosphate isomerase (EC 5.3.1.1) (TIM)

UMP-CMP kinase (Cytidylate kinase) (Deoxycytidylate kinase) (Cytidine monophosphate kinase) Phosphoglycerate kinase, testis specific (EC 2.7.2.3) Phosphoglycerate mutase 1 (Phosphoglycerate mutase isozyme B) (PGAM-B) (BPG-dependent PGAM 1) Adenine phosphoribosyltransferase (APRT)

5.57 6.79

6.51

5.57

8.35

6.08

41.5

21.0

55.5 57.0

24477.24 6.12

19545.75 5.76

25581.33 5.62 6.20 26789.78 6.42 7.50 26402.21 5.73 39152.59 6.54-7.82

40675.24 6.44

28718.32 5.53

28.0 16.6 24.6 24.8 25.0 37.8

35.2

21.0

29.9 109 43.0

26.7

20.5

28514.47 6.54-7.50 26.2

44423.22 8.55

57686.60 7.30 57649.61 7.60 7.85 22165.33 5.70

5.88 32809.01 5.96 6.22 116979.69 6.30 6.53 48332.63 6.10

6.07

6.17

6.21

7.52

5.68

6.69 7.40

7.03

19.8 52.0 53.0 21.2 21.6 44.0 31.2 43.0 43.0 26.5 33.6

18632.25 5.95 56550.80 5.68 5.83 25655.33 6.43 7.10 42712.23 4.42 5.38 5.51 5.56, 5.60 4.90 34908.12 6.09, 6.29

6.21 5.57

112.0 110.0 32.2

scorec

no. of spot pI

20 19

18

7

15

5

18 24 12

19

12

19

27

12

33 34

24

29

19

19 35

34

11

41

3 1 2 2 6

2 1 2

359e 207e 250e 287e 339e

152e 265e

3 1 1 1 1 1 1 1 2 2 1 4

187e 247e 184e 247e

159e 120e 110e 229e 212e 187e

81 91e

1

196

84

1

1

113e

275e

2

191

5.74 6.68-7.40

5.63 6.18 6.67-7.40

6.34

5.55

5.70 5.77 5.78 6.08 5.94 6.22 6.05

7.40-8.10 5.76 6.22-6.65

6.05

6.11 6.15 6.89 5.15 5.40 5.56 5.71 6.05 6.20 5.53 6.28-7.12

5.94 5.67

8.02

5.49

6.11

25.6 39.0

28.1 16.6 25.0

35.7

21.6

12.2 21.0 22.8 27.2 30.3 108.0 44.5

42.0 37.2 26.6

26.2

21.0 20.3 16.7 27.3 32.0 44.5, 54.8 45.2 34.2 33.8 34.3 52.0-59.4

20.0 54.0

50.5

33.0

110.0

MW (kDa)

9 21

14

8

17

4

14 19 16

17

10

19

17

12

19 18

14

23

9

11 28

26

10

45

matches

observedb

observedb

matches

astrocytes

neurons

MW (kDa)

58826.46 6.90-8.70 49.0

6.32

Pyruvate kinase, M1 isozyme (EC 2.7.1.40) Pyruvate kinase, M2 isozyme (EC 2.7.1.40)

pI

9.22

PDXK_RAT

Pyridoxal kinase (EC 2.7.1.35) (Pyridoxine kinase)

MW (Da)

Metabolic proteins 6.36 116448.97 6.19 6.42 6.41 38937.09 5.48

pI

O35331

Ogdh protein [2-oxoglutarate dehydrogenase]

protein name (synonym)

5.33

Q91WP2


theoreticala

ODPB_MOUSE Pyruvate dehydrogenase E1 component β subunit, mitochondrial [precursor] (EC 1.2.4.1) (PDHE1-B). P15999 ATPA_RAT ATP synthase R chain, mitochondrial [Precursor] [Fragment] (EC 3.6.3.14) P31399 ATPQ_RAT ATP synthase D chain, mitochondrial (EC 3.6.3.14) P50517 VAB2_MOUSE Vacuolar ATP synthase subunit B, brain isoform (EC 3.6.3.14) (V-ATPase B2 subunit) CAA39599 H(+)-transporting ATP synthase [Rattus norvegicus] P07335 KCRB_RAT Creatine kinase, B chain (EC 2.7.3.2) (B-CK)

Q9D051

Q91WP2

no.

Table 1 (Continued)

67 324e

123e

156e

201e

91e

82 242e

256e

183e

114e

1 3

3

2

1

1

1 1 1

2

2

3

4

309e 187e

1

1 7

2

6

2

1 1

1

1

1

no. of spot

104

202e

147e

85

207e

71

95 217e

265e

83e

259

scorec


Q8BTY3 ITPA_MOUSE DDH2_MOUSE

PA1G_RAT

CLPP_MOUSE

Q8BTY3 Q9D892 Q99LD8

O35263

O88696

ODO2_RAT

Q01205

UCRI_RAT

ODP2_RAT

P08461

P20788

AATC_RAT

P13221

Q920L2

ATPD_RAT Q8CI65

P35434 Q8CI65

Q920L2

ATPB_RAT

P10719

NUAM_MOUSE

COXE_RAT

P10818

Q91VD9

COXB_RAT

P12075

Q8VC72

COXA_RAT

P11240

Q8VC72

ENOG_RAT

P07323

Q80WE0

FRIH_RAT ENOA_RAT

P19132 P04764

Q80WE0

Q9CQ60

Q9CQ60

SCOT_MOUSE

Q99JB2 Q99KI0

Q99JB2 Q99KI0

Q9D0K2

TKT_RAT CATD_RAT Q8CHX2

P50137 P24268 Q8CHX2

Aspartate aminotransferase, cytoplasmic (EC 2.6.1.1) (Glutamate oxaloacetate transaminase-1) Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex [Fragment] Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial [Precursor] (EC 2.3.1.61) (E2) (E2K) Succinyl-CoA:3-ketoacid-coenzyme A transferase 1, mitochondrial [precursor] (EC 2.8.3.5) (Scot-S) NADH dehydrogenase 1 R subcomplex 10-like protein NADH dehydrogenase (Ubiquinone) Fe-S protein 8 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial [Precursor] (Complex I-75Kd) (CI-75Kd) Flavoprotein subunit of succinate-ubiquinone reductase Ubiquinol-cytochrome c reductase iron-sulfur subunit, mitochondrial [Precursor] [Fragment] (RISP) Inorganic pyrophosphatase Inosine triphosphate pyrophosphatase NG,NG-dimethylarginine dimethylaminohydrolase 2 (EC 3.5.3.18) (Dimethylargininase 2) (DDAHII). Platelet-activating factor acetylhydrolase IB γ subunit (PAFAH γ subunit) (PAF-AH R 1) Putative ATP-dependent Clp protease proteolytic subunit, mitochondrial precursor (Endopeptidase Clp)

ATP synthase δ chain, mitochondrial [precursor] Atp5b protein [Fragment]

γ enolase (EC 4.2.1.11) (2-phospho-D- glycerate hydro-lyase) (Neural enolase) (NSE) (Enolase 2) Cytochrome c oxidase polypeptide Va, mitochondrial [Precursor] (EC 1.9.3.1) Cytochrome c oxidase polypeptide Vb, mitochondrial [Precursor] (EC 1.9.3.1) Cytochrome c oxidase polypeptide VIaliver, mitochondrial [Precursor] (EC 1.9.3.1) ATP synthase β chain, mitochondrial [Precursor]

Stomatin-like protein 2 Similar to mitochondrial aconitase (Nuclear aco2 gene) 6-phosphogluconolactonase (1110030K05Rik protein (RIKEN cDNA 1110030K05 gene)) Ferritin heavy chain (Ferritin H subunit) R enolase (EC 4.2.1.11) (2-phospho-D- glycerate hydro-lyase) (Nonneural enolase) (NNE) (Enolase 1)

Transketolase (EC 2.2.1.1) (TK) Cathepsin D [Precursor] (EC 3.4.23.5) ATPase, H+ transporting, lysosomal 70kD, V1 subunit A, isoform 1

7.05

6.42

5.76 5.60 5.66

8.90

6.75

5.51

5.19

7.14

8.73

8.17

5.70

6.30

5.16 5.24

5.18

9.30

7.68

6.08

5.03

5.85 5.84

5.55

8.95 8.08

7.22 6.66 5.42

29800.49

25863.44

32565.03 21897.02 29645.83

27688.62

71615.21

79748.74

18573.15

40544.11

55988.57

47412.70

58764.14

46197.42

17595.07 56666.87

56353.55

12301.04

13914.92

16129.53

47009.34

20995.47 47069.72

27254.43

38384.91 85463.51

67643.64 44680.69 68326.08

5.66

6.60

5.41 5.46 5.61

7.45

6.19, 6.29

5.98

5.25

6.16

7.30, 7.60

5.91

5.70

5.11-5.55 4.93 5.10 4.20 5.12 5.42 7.50

6.64

5.43

5.50 5.95-6.25 5.50 5.75 5.81 5.46 4.99-5.17 5.01

5.52

7.88 5.60 5.53 5.51 5.55 5.90 7.68, 7.90

25.8

25.7

33.0 21.4 28.4

24.0

68.0

57.8

21.4

36.9

52.0

50.0

64.2

43.0-47.0 32.2 23.8 13.8 27.3 23.7 39.6

11.6

12.0

19.2 42.0-48.0 37.0 48.0 48.5 8.5 34.0-46.2 11.4

26.6

65.0 41.0 68.0 38.6 25.8 37.9 81.0

14

11

13 6 15

11

22

13

9

13

17

23

15

29

4 16

26

8

12

10

27

17 21

7

18 30

8 13 24

1 1 8

5

152e 164e

280e

75

1

1

1 1 1

98e 77e 71e 82

1

2

235e 71

1

1

75e 67

1

95

2

1

206e

74

1

1

276e 94

1 2

5 63 144e

1

302e

1

73

96

89

1

1 2

216e 302e

181e

1 1 3

75 79e 176e

5.72 5.99 5.86 5.77-5.90 6.02-6.25

5.52

5.87 7.07

7.37 5.60, 5.76 5.53 5.51

18.0 43.0 35.7 49.0-49.8 49.0

27.2

38.8 87.0

68.6 42.1 70.5 39.6

11 19

10

14 22

21 17 27

90 133e

91e

189e 149

172e 148e 180e

1 7

1

1 1

1 2 2


research articles


778

CATB_RAT

MPPB_RAT

MEPD_RAT

ODPA_RAT

PYC_RAT KPR1_HUMAN

Q8BHN3 Q8BL86

VATF_MOUSE

FABH_RAT FRIL_RAT Q924S5

SCB1_MOUSE

SCB2_MOUSE

ASSY_RAT

SERA_RAT UGDH_RAT DHE3_RAT

DHB8_MOUSE MMSA_RAT

LDHA_RAT

Q9JLJ3 NIT1_RAT Q9JHW2 DDH1_RAT

NUFM_RAT

NB6M_MOUSE

Q923D2

Q03346

P24155

P26284

P52873 P60891

Q8BHN3 Q8BL86

Q9D1K2

P07483 P02793 Q924S5

Q9Z2I9

Q9Z2I8

P09034

O08651 O70199 P10860

P50171 Q02253

P04642

Q9JLJ3 Q7TQ94 Q9JHW2 O08557

Q63362

Q9ERS2

Q923D2


P00787

no.

Table 1 (Continued)

Mitochondrial processing peptidase β subunit, mitochondrial precursor (β-MPP) (P-52) Thimet oligopeptidase (EC 3.4.24.15) (Endo-oligopeptidase A) (Endopeptidase 24.15) (PZ-peptidase) Pyruvate dehydrogenase E1 component R subunit, somatic form, mitochondrial [Precursor] (EC 1.2.4.1) Pyruvate carboxylase, mitochondrial [precursor] Ribose-phosphate pyrophosphokinase I (Phosphoribosyl pyrophosphate synthetase I) (PRS-I) (PPRibP). R glucosidase 2 Hypothetical Metallo-β-lactamase superfamily containing protein [96.42% identity to|Q8IY16, Similar to metallo-β-lactamase superfamily protein] Vacuolar ATP synthase subunit F (EC 3.6.3.14) (V-ATPase F subunit) (Vacuolar proton pump F subunit) Fatty acid-binding protein, heart (H-FABP) Ferritin light chain (Ferritin L subunit) Lon [96.84% identity to Q8CGK3, Mitochondrial ATP-dependent protease Lon] Succinyl-CoA ligase [ADP-forming] β-chain, mitochondrial [Precursor] [Fragment] (SCS-βA) Succinyl-CoA ligase [GDP-forming] β-chain, mitochondrial [Precursor] [Fragment] (SCS-βG) (GTP-specific succinyl-CoA synthetase β subunit) Argininosuccinate synthase (EC 6.3.4.5) (Citrulline-aspartate ligase) D-3-phosphoglycerate dehydrogenase (3-PGDH) UDP-glucose 6-dehydrogenase (UDPGDH) Glutamate dehydrogenase, mitochondrial [Precursor] Estradiol 17-β-dehydrogenase 8 (17-β-HSD 8) Methylmalonate-semialdehyde dehydrogenase [acylating], mitochondrial [Precursor] (MMSDH) L-lactate dehydrogenase A chain (EC 1.1.1.27) (LDH-A) (LDH muscle subunit) (LDH-M) 4-trimethylaminobutyraldehyde dehydrogenase Nitrilase homologue 1 Nit protein 2 NG,NG-dimethylarginine dimethylaminohydrolase 1 (Dimethylargininase 1) (DDAHI) (DDAH-1) NADH-ubiquinone oxidoreductase 13 kDa-B subunit (Complex I-13Kd-B) (CI-13Kd-B) NADH-ubiquinone oxidoreductase B16.6 subunit (Complex I-B16.6) (CI-B16.6) (GRIM-19) Blvrb protein (Biliverdin reductase B) (Flavin reductase (NADPH))

Cathepsin B [precursor] (Cathepsin B1) (RSG-2)


MW (Da)

pI

Journal of Proteome Research • Vol. 4, No. 3, 2005 6.49

9.47

7.07

6.57 5.92 6.44 5.76

8.45

6.10 8.47

6.28 7.48 8.05

7.63

5.75

5.65

5.92 5.98 6.17

5.52

5.67 6.36

6.25 6.56

8.49

5.54

5.36

22197.33

16728.40

13280.59

53652.72 32090.68 30501.76 31294.82

36450.50

26645.39 57807.60

56493.42 54892.01 61427.94

46496.31

43770.30

46244.47

14643.52 20674.36 105792.52

13370.27

106911.11 31206.39

129689.61 34703.04

43212.55

78183.08

5.84

5.67 6.04 5.93

5.42

5.71-5.79 6.42

6.19, 6.45 7.10

7.28

5.74

Metabolic proteins 37469.96 5.33 5.29 6.38 54318.21 5.75

pI

theoreticala

42.5

11.5 17.7 107.0

10.5

107.0 28.5

132.0 32.3

41.5

79.0

23.8 26.6 46.5

22

15 18 26

15

27 10

39 14

16

16

27

15

matches

scorec

no. of spot pI

99

1

1 1 1

1

200e 125 177e 136

3 1

2 1

1

1

1

2

113 102e

201 102e

91

71

179

214e

6.54

6.11

6.39

5.92 5.84 6.64 5.74

8.51

6.04 7.66

6.28 7.42 6.66, 6.79

7.40

5.79

26.6

70.0

11.2

50.1 29.7 28.9 36.2

34.0

30.2 53.4

54.7 55.5 50.3

43.0

42.9

MW (kDa)

11

7

8

18 12 11 12

21

9 19

18 17 28

10

17

matches

observedb

observedb

MW (kDa)

astrocytes

neurons

1 1

80 85e

1

1 1 1 1

228e 69 93 76 65

1

111

62 82

1 1

1 1 2 132 358e

1 171e

1

no. of spot

81

92

scorec


Q8BUM1 RSP4_RAT

ROK_RAT

O88612 MAT3_RAT

P61980

O88612 P43244

Q811L7

Q811L7

Q8BUM1 P38983

Q9D6G1 Q8CJ71

Q9D6G1 Q8CJ71

RBM3_MOUSE

SYG_MOUSE

Q9CZD3

O89086

EF2_RAT Q8BFR5 FUB2_RAT

P05197 Q8BFR5 Q99PF5

DD48_MOUSE

DDX3_MOUSE

Q62167

Q91VC3

GALE_RAT Q9JKB7 Q9CZS4

P18645 Q9JKB7 Q9CZS4

PCB2_MOUSE

CPT2_RAT

P18886

Q61990

PNPH_MOUSE

P23492

PCB1_MOUSE

Q99P44 DCUP_RAT

Q99P44 P32362

P60335

Q99K88 ETFA_MOUSE

Q99K88 Q99LC5

RT22_MOUSE

Q6PHC1 AMRP_RAT

Q6PHC1 Q99068

Q9CXW2

ALDR_RAT

P07943

Q9Z130

ALFA_RAT

P05065

Q9Z130

ECH1_RAT

Q62651

Poly(rC)-binding protein 2 (R-CP2) (Putative heterogeneous nuclear ribonucleoprotein X) (CTBP) Probable ATP-dependent helicase DDX48 (DEAD-box protein 48) Putative RNA-binding protein 3 (RNA binding motif protein 3) TAR DNA binding protein homolog 40S ribosomal protein SA (p40) (34/67 kDa laminin receptor) Heterogeneous nuclear ribonucleoprotein K (dC-stretch binding protein) (CSBP) High mobility group protein Matrin 3

JKTBP (Heterogeneous nuclear ribonucleoprotein D-like) Mitochondrial 28S ribosomal protein S22 (MRP-S22) Poly(rC)-binding protein 1 (R-CP1) (hnRNP-E1).

DEAD-box protein 3 (DEAD-box RNA helicase DEAD3) Elongation factor 2 (EF-2) Elongation factor TU Far upstream element binding protein 2 (FUSE binding protein 2) (KSRP) (MAP2 RNA trans-acting protein 1) Glycyl-tRNA synthetase (GlycinestRNA ligase GlyRS) Heterogeneous nuclear ribonucleoprotein A/B Heterogeneous nuclear ribonucleoprotein A2/B1/B0 Heterogeneous nuclear ribonucleoprotein H1

δ3,5-δ2,4-dienoyl-CoA isomerase, mitochondrial [Precursor] Fructose-bisphosphate aldolase A (EC 4.1.2.13) (Muscle-type aldolase) Aldose reductase (EC 1.1.1.21) (Aldehyde reductase) Eno1 protein [R enolase] R-2-macroglobulin receptor-associated protein [Precursor] [Fragment] (R-2-MRAP) (RAP) Acat2 protein Electron-transfer flavoprotein R-subunit, mitochondrial [Precursor] (R-ETF) Leucine aminopeptidase 3 (EC 3.4.11.1) Uroporphyrinogen decarboxylase [Fragment] (EC 4.1.1.37) (URO-D) (UPD) Purine nucleoside phosphorylase (EC 2.4.2.1) (Inosine phosphorylase) (PNP) Carnitine O-palmitoyltransferase II, mitochondrial [Precursor] (EC 2.3.1.21) (CPT II) UDP-glucose 4-epimerase (Galactowaldenase) Guanine deaminase Hypothetical protein [65.98% identity to Q9BVV7, HSPC154 protein] 23

5.26 5.82

5.69

6.55 4.80

6.84

6.30

6.33

6.66

8.63

6.85

5.89

6.07 8.97

6.24

6.42 7.23 6.38

24923.66 94447.02

48510.49

33634.12 32824.05

16604.69

46839.98

38221.68

37497.81

41192.11

33558.79

49199.44

29922.29 37402.67

81877.77

95152.82 49508.35 74226.45

6.21 5.75-5.82

5.54

5.85-6.30 4.60

6.92

24.9 130.0

61.8

41.0 34.0

15.4

44.8

38.8 39.7

7.00 6.38 6.29

39.0

37.1

22.8 29.0 49.0-46.0 34.9

29.8 32.8, 33.3

74.0

95.0 42.9 81.0

6.35

6.31

6.39 5.53 5.79-5.93 6.20

5.90 7.65

5.93, 5.96

7.20 6.50 6.50-6.90

14 20

21

12 14

11

16

9

16

12

8

20

15 17

12

34 21 30

1 1 3 2 1 2 6

319e

97e 147e 174e 203e

1

224e 80 142e

1 3

4 1

152e 120e

1

73

1

142e

2

139e

1

1

115e

70

1

79e

153e 210e

1

163e

5.95, 6.16

6.72

6.19

6.26

6.24, 6.66

6.18

6.15

5.95

6.40 7.20, 8.57

5.91

6.65, 6.83

6.68, 6.75

6.75

73.8

Nucleic acid binding proteins 6.73 72970.20 7.18

6.64

6.30

6.23, 6.37 6.06

6.59, 6.78 67.8, 7.08

5.74 6.71

6.26

7.08

6.49

6.02 5.57 7.70

74110.34

32277.06

56141.32 40452.69

38147.17 35039.47

38299.66 41732.33

35666.08

39220.75

36171.67

38224.81 50900.97 27911.08

7.66 5.48 9.97

6.89

5.78

7.61 6.35

7.63 8.62

5.86 6.90

6.28

8.40

8.14

42.5

15.4

46.3

41.1

40.0

39.0

35.8

21.6

41.0 33.0

76.0

85.0

97.0

76.0

38.0 46.7 30.1

70.0

29.9

55.7 38.0

38.2 31.0

38.8 40.0

35.3

33.0

31.1

13

10

13

14

20

16

12

9

9 14

15

34 18 29

19

22 17 9

19

16

30 15

11 8

12 10

13

22

13

2 1 1

230e 82 164e

2

1 120e

1 94e

1

2

1

1

1

1 2

1

2 1 2

1

1 1 1

62

69

203e

114

98e

96e

71 144e

83e

127 219e

319e

223e

154 105 75

1

2 2

89e 122e

104

1 1

1

2

1

73 122e

92

178

67


research articles


780

Q8VIJ6 RUV1_RAT

SYS_MOUSE

Q810V5

SFR3_HUMAN

PUR_MOUSE

RLA0_RAT

CRKL_MOUSE DOPD_RAT Q8K1G4 SUI1_MOUSE

IF4H_MOUSE

FUB1_MOUSE

ROL_MOUSE

UBF1_RAT

CRK_RAT

Q9Z2R7 Q6TNG5 Q76M56

PRS7_RAT O00487

PSA1_RAT

PSA2_RAT

PSA3_RAT

PSA6_HUMAN

PSB3_RAT

P26638

Q810V5

P23152

P42669

P19945

P47941 P80254 Q8K1G4 P48024

Q9WUK2

Q91WJ8

Q8R081

P25977

Q63768

Q9Z2R7 Q6TNG5 Q76M56

Q63347 O00487

P18420

P17220

P18422

P60900

P40112


Q8VIJ6 P60123

no.

Table 1 (Continued)

MW (Da)

pI

MW (kDa)

11.64

5.46

5.95

19329.60

30308.77

58257.52

5.80 5.89 6.03 5.94

5.75

6.01

20.2 20.1 19.7 39.3

30.5

60.5

Nucleic acid binding proteins 9.45 75441.66 7.33 100.0 6.02 50213.98 6.33 49.6

pI

Transcriptional activator protein PUR-R 6.07 34883.73 (Purine-rich single-stranded DNAbinding protein R). 60S acidic ribosomal protein P0 60S 5.91 34215.47 acidic ribosomal protein P0 (L10E) Crk-like protein 6.26 33817.04 D-dopachrome tautomerase (EC 5.3.3.-) 6.15 13002.08 Elongation factor-2 [Fragment] 6.04 33878.59 Eukaryotic translation initiation factor 1 (eIF1) 6.90 12746.52 (Protein translation factor SUI1 homolog) Eukaryotic translation initiation factor 4H (eIF-4H) 6.67 27341.01 (Williams-Beuren syndrome chromosome region 1 protein homolog) Far upstream element binding protein 1 7.73 68539.59 (FUSE binding protein 1) (FBP) Heterogeneous nuclear ribonucleoprotein 6.65 60123.22 L (hnRNP L) Nucleolar transcription factor 1 (Upstream 5.63 89436.87 binding factor 1) (UBF-1). Proto-oncogene C-crk (P38) 5.39 33844.72 (Adapter molecule crk). Replication factor C [Fragment] 9.40 123246.96 Staufen 9.87 48740.13 Transcription factor BTF 3 Basic 6.85 17699.04 transcription factor 3) Ubiquitin and proteasomal proteins 26S protease regulatory subunit 7 5.60 48443.59 5.83 45.2 26S proteasome-associated pad1 6.06 34577.04 6.02 32.8 homolog Proteasome subunit R type 1 (Proteasome 6.14 29517.50 6.25 29.2 component C2) (Macropain subunit C2) Proteasome subunit R type 2 (Proteasome 7.12 25795.41 7.08 23.0 component C3) (Macropain subunit C3) Proteasome subunit R type 3 (Proteasome 5.29 28288.05 6.41 11.4 component C8) (Macropain subunit C8) Proteasome subunit R type 6 (Proteasome 6.35 27399.45 6.28 25.4 ι chain) (Macropain iota chain) (27 kDa prosomal protein) Proteasome subunit β type 3 (Proteasome 6.15 22964.88 6.08 23.6 θ chain) (Proteasome chain 13) (Proteasome component C10-II)

PTB-associated splicing factor RuvB-like 1 (EC 3.6.1.-) (49-kDa TATA box-binding protein-interacting protein) (TIP49a) (DNA helicase p50) Seryl-tRNA synthetase (EC 6.1.1.11) (SerinestRNA ligase) (SerRS) Similar to no on or off transient A [Fragment] Splicing factor, arginine/serine-rich 3 (Pre-mRNA splicing factor SRP20) (X16 protein)


theoreticala scorec

no. of spot pI


16

8

14

13

36 20

10

7

8

20

15 24

1 1 1 1 1

135e 89e 161e 279e

1

93e

137e

3

1 1

1

116e

294 156e

1

78e

1 1

92

80e 259e

6.05

6.20

6.39

6.32-6.73

6.18

5.81 5.99

7.00 5.68 7.00

5.62

6.33 6.64

6.81

6.80 7.47

6.15

6.17 6.16 6.23 6.76

5.73

24.0

25.9

11.2

23.2

29.8

46.6 33.1

85.2 57.3 17.3

31.1

73.0 77.5

69.1

74.2 71.5

28.9

38.0 11.5 13.0 11.8

35.8

MW (kDa)

14

17

12

9

19

28 17

22 9 12

11

18

20

15

9

14 13 13 6

14

matches

observedb

observedb

matches

astrocytes

neurons

124

216e

66

61

1

1

1

2

1

1 1

211e 107 225e

1 1 1

1

2

1

2

1

1 1 3 1

1

no. of spot

62 70 105

83

74

109

87

86

83 117 115e 105e

169e

scorec


143Z_MOUSE

BNT1_RAT KAD1_RAT

NDKA_RAT

P35215

Q9Z1N4 P39069

Q05982

O88544

O88544

PP1B_HUMAN

CRTC_RAT

P18418

P62140

Q9EQX9 Q9ES53

Q9EQX9 Q9ES53

GDIC_MOUSE

PRSX_MOUSE

P62334

Q61598

PRS8_MOUSE

P62196

PEBP_RAT

UBIQ_HUMAN UBL1_MOUSE

P02248 Q9R0P9

P31044

UBQ1_MOUSE

Q8R317

NDKB_RAT

PSB2_RAT

P40307

P19804

PSE3_MOUSE

P61290

PPAC_RAT

PSE1_MOUSE

P97371

P41498

PSD7_MOUSE

P26516

GRB2_RAT

PSB7_RAT

Q9JHW0

P62994

PSB4_RAT

P34067

Calreticulin [Precursor] (CRP55) (Calregulin) (HACBP) (ERp60) (CALBP) (Calcium-binding protein 3) (ABP3) COP9 complex subunit 4 (COP9 (Constitutive PHOTOMORPHOGENIC), subunit 4) (Arabidopsis) Growth factor receptor-bound protein 2 (GRB2 adapter protein) (SH2/SH3 adapter GRB2) (ASH protein) Low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) (PTPase) Nucleoside diphosphate kinase B (EC 2.7.4.6) (NDK B) (NDP kinase B) (P18) Phosphatidylethanolamine-binding protein (PEBP) (HCNPpp) (23 kDa morphine-binding protein) (P23K) Rab GDP dissociation inhibitor β-2 (Rab GDI β-2) Serine/threonine protein phosphatase PP1-β catalytic subunit (EC 3.1.3.16) (PP-1B) 14-3-3 protein ζ/δ (Protein kinase C inhibitor protein-1) (KCIP-1) 3′(2′),5′-bisphosphate nucleotidase 1 (EC Adenylate kinase isoenzyme 1 (EC 2.7.4.3) (ATP-AMP transphosphorylase) (AK1) (Myokinase) Nucleoside diphosphate kinase A (EC 2.7 4.6) (NDK A)

Proteasome subunit β type 4 [precursor] (Proteasome β chain) (Macropain β chain) Proteasome subunit β type 7 [precursor] (Proteasome subunit Z) (Macropain chain Z) 26S proteasome non-ATPase regulatory subunit 7 (26S proteasome regulatory subunit S12) (Mov34 protein) Proteasome activator complex subunit 1 (Proteasome activator 28-R subunit) (PA28R) (REG-R) Proteasome activator complex subunit 3 (Proteasome activator 28-γ subunit) (PA28γ) (PA28 g) Proteasome subunit β type 2 (Proteasome component C7-I) (Macropain subunit C7-I) Ubiquilin 1 (Protein linking IAP with cytoskeleton-1) Ubiquitin Ubiquitin carboxyl-terminal hydrolase isozyme L1 (EC 3.4.19.12) (UCH-L1) (Ubiquitin thiolesterase L1) (Neuron cytoplasmic protein 9.5) (PGP 9.5) (PGP9.5) 26S protease regulatory subunit 8 (Proteasome subunit p45) (Proteasome 26S subunit ATPase 5) (mSUG1). 26S protease regulatory subunit S10B (Proteasome subunit p42) (Proteasome 26S subunit ATPase 6) Bendless protein UFD1 [Ubiquitin fusion degradation protein 1 homolog] 17123.79 34485.37

44172.99

45626.01

8564.84 24838.26

61976.39

22912.38

29506.06

28672.94

36539.82

29927.46

29197.29

6.85 5.43 5.44 5.32-5.44

4.74

6.75

5.69

5.58

6.90

5.99

5.84

5.96

5.58 7.72

4.73

5.84

5.93

5.48

6.91

6.10

5.89

5.57

17192.74

33174.03 21601.72

27771.14

37186.83

50537.13

20670.20

17282.98

18020.42

25206.35

46284.81

5.80

5.70 7.70

5.10

15.4

39.9 20.9

23.0

35.3

16.5 45.1

5.53 5.96 5.87

20.0

16.3

16.2

23.7

40.0

60.0

8.0 16.6 17.8 25.7-24.6

65.5

21.2

29.5

27.8

36.0

27.3

22.6

5.44

7.10

5.84

5.85

5.60

Signaling proteins 4.33 47995.47 4.30

6.13 6.27

7.09

7.11

6.56 5.14

4.86

6.96

5.69

5.73

6.29

8.14

6.45

15

17 12

8

28

25

15

19

13

20

21

22

9 16

16

6

12

9

13

15

16

1 1

136e 120e

107

107 140

1

2 1

1

1

388e 63

1

2

185e 153

1

1 164

76

1

1

152e 188e

1

213e

1 5

1

94e 92 105

1

61

1

1

144e

77

1

86

5.84

5.87, 5.92

5.46

6.80

5.85

5.83

4.15 4.39 4.35 5.62

5.85 6.32

7.09

6.98

5.95

5.83

36.2

46.6

20.5

15.7

16.4

24.1

38.0 46.7 65.0 40.8

14.7 39.0

41.1

44.7

27.8

23.0

16

26

12

13

13

18

16

15

12 11

16

19

13

11

1 1

119e 64

1

116e

116

220e

1

2

1

1

185e

89

1

1 152

102

3

1

112e

100

1

1

1

191e

117e

71


research articles


782

Q8CDT0 Q8BND1 SUH_MOUSE

GDIR_MOUSE

PP1A_MOUSE

PAK2_RAT

STN1_RAT

CAP1_MOUSE

ANX1_RAT

ANX2_RAT

ANX3_RAT

ANX4_RAT

Q8VIN2 CALM_HUMAN CLI4_MOUSE

RAN_HUMAN

GBLP_HUMAN

CALD_RAT NU54_RAT Q9QV93 PPI1_RAT

Q80Z29 PTD4_MOUSE

O88767 Q91XM8

DPY2_RAT

Q99PT1

P62137

Q64303

AAH59848 P13668

P40124

P07150

Q07936

P14669

P55260

Q8VIN2 P62158 Q9QYB1

P17080

P63244

Q62736 P70582 Q9QV93 P16446

Q80Z29 Q9CZ30

O88767 Q91XM8

P47942


Q8CDT0 Q8BND1 P31266

no.

Table 1 (Continued)


Dihydropyrimidinase related protein-2 (DRP-2) (Collapsin response mediator protein 2) (CRMP-2)

CAP1 protein [Contraception associated protein 1] Collapsin response mediator protein 4

Annexin A2 (Annexin II) (Lipocortin II (P36) (Protein I) Annexin A3 (Annexin III) (Lipocortin III) (Placental anticoagulant protein III) (PAP-III) Annexin A4 (Annexin IV) (Lipocortin IV) (36 kDa zymogen granule membraneassociated protein) Annexin VII Calmodulin (CaM) Chloride intracellular channel protein 4 (mc3s5/mtCLIC) GTP-binding nuclear protein Ran (TC4) (Ran GTPase) (Androgen receptorassociated protein 24) Guanine nucleotide-binding protein β subunit 2-like 1 (Receptor of activated protein kinase C 1) (RACK1) Nonmuscle caldesmon (CDM) (L-caldesmon) Nucleoporin p54 (54 kDa nucleoporin) P93 calcium-binding protein [Fragment] Phosphatidylinositol transfer protein R isoform (PtdIns transfer protein R) (PtdInsTP) (PI-TP-R) Pre-B-cell colony-enhancing factor Putative GTP-binding protein PTD004 homolog

IK cytokine [Fragment] Protein kinase Recombining binding protein suppressor of hairless (J kappa-recombination signal binding protein) Rho GDP-dissociation inhibitor 1 (Rho GDI 1) (GDI-1) Serine/threonine protein phosphatase PP1-R catalytic subunit (EC 3.1.3.16) (PP-1A) Serine/threonine-protein kinase PAK 2 (p21activated kinase 2) (PAK-2) (γ-PAK) (p62-PAK) Sept5 protein Stathmin (Phosphoprotein p19) (pp19) (Oncoprotein 18) Adenylyl cyclase-associated protein 1 (CAP 1) Annexin A1 (Annexin I) (Lipocortin I) (Calpactin II) (Chromobindin 9) (P35)


60584.05 55744.84 2519.79 31776.23

35076.73

24423.11

50079.39 16706.39 28729.04

35717.48

36322.19

38547.05

38698.28

51443.67

41411.33 17157.29

57960.35

37540.09

23407.41

6.17 5.47, 5.65

5.74

5.94

5.20

Signaling proteins 44568.69 6.36 76350.45 5.81 58537.41 5.37

pI

6.69 55437.90 7.64 44729.54 Miscellaneous proteins 5.76 19974.17 5.75, 6.05 6.04 61967.31 5.65-5.84 6.07-6.25 5.95 62277.57 5.56 5.61 5.88-6.60 5.58, 5.65

6.34 6.53 4.56 5.98

7.60

7.01

5.91 4.09 5.44

5.32

6.04

7.53

7.13

7.30

5.89 5.77

5.57

5.94

5.12

6.74 5.74 8.43

MW (Da)

theoreticala pI

21.4 63.0 59.0 57.1 56.0 61.0-72.0 68.5

40.0 16.0

58.0

35.8

24.8

77.0 79.0 100.3

41

16 26

21 4

20

21

11

11 12 16

matches

scorec

no. of spot pI

2 6 13

393e 161e 403e

1 2

1

159 74e

1

181e

1

1 1 1

199

64

64 63 60

5.95, 6.10

6.01 6.02, 6.18

6.80 6.20

6.00, 6.20 6.56 3.0.96 6.10

7.03

6.88

5.78 3.70 6.12

5.48

5.94

5.66 5.84 6.33, 6.67 6.76

6.80,7.09

65.0

22.2 61.0

54.0 28.0

76.0 58.0 15.6 32.0

30.0

23.7

45.0 15.5 24.5

31.4

31.6

25.0 25.3 36.7 10.1

55.0

MW (kDa)

25

19 19

16 9

29 14 3 20

17

8

21 5 10

14

25

13

23

8

matches

observedb

observedb

MW (kDa)

astrocytes

neurons

1 1 1

161e 418e 108e

242e

184e 141

105 90

214 78 82e 138

147

74

2

1 4

1 1

4 1 1 1

1

1

1 1 1

4

105 85e 63

2 427e

no. of spot

128e

scorec


ARF1_HUMAN Q8VIF7 Q91Y81 Q9D402

ERH_HUMAN Q8C8W5 GLMB_RAT Q8K4F7 MED2_MOUSE Q8R5L1

Q14259 Q8C8W5 Q63228 Q8K4F7 Q9ERE7 Q8R5L1

P32889 Q8VIF7 Q91Y81 Q9D402

DPY5_RAT

Q9JHU0

TCTP_MOUSE

DPY4_RAT

Q62951

P14701

DPY1_RAT

Q62950

LEG1_RAT

CTP1_RAT

Q9Z2F5

P11762

CBX3_MOUSE

P23198

LEG3_RAT

BAF_RAT

Q9R1T1

P08699

POR1_RAT

Q9Z2L0

SN25_RAT

Q8C1B7

AAH46532 Q8C1B7

P60881

PHB_MOUSE

P24142

SRN1_MOUSE Q91YN9

PD6I_MOUSE

Q9WU78

Q9CZC8 Q91YN9

ER29_RAT FABE_RAT HNT1_MOUSE

P52555 P55053 P70349

Voltage-dependent anion-selective channel protein 1 (VDAC-1) (rVDAC1) Barrier-to-autointegration factor (LAP2binding protein 1). Chromobox protein homologue 3 (Heterochromatin protein 1 homologue γ) (HP1 γ) (Modifier 2 protein) C-terminal binding protein 1 (CtBP1) (C-terminal binding protein 3) (CtBP3) (BARS-50) Dihydropyrimidinase related protein-1 (DRP-1) (Collapsin response mediator protein 1) (CRMP-1) Dihydropyrimidinase related protein-4 [Fragment] (DRP-4) (Collapsin response mediator protein 3) (CRMP-3) Dihydropyrimidinase related protein-5 (DRP-5) (ULIP6 protein) Enhancer of rudimentary homolog Ethanol decreased 4 [Fragment] Glia maturation factor β (GMF-β) Histidine triad protein member 5 Mesoderm development candidate 2 P32-RACK (Complement component 1, q subcomponent binding protein) Secernin 1 Similar to BCL2-associated athanogene 2 (BAG-family molecular chaperone regulator-2 homolog) Synaptosomal-associated protein 25 (SNAP-25) (Super protein) (SUP) Galectin-3 (Galactose-specific lectin 3) (35 kDa lectin) Galectin-1 (β-galactoside-binding lectin L-14-I) (Lactose-binding lectin 1) (S-Lac lectin 1) (Galaptin) Translationally controlled tumor protein (TCTP) (p23) (21 kDa polypeptide) (p21) (Lens epithelial protein) ADP-ribosylation factor 1 Selenium-binding protein Vascular endothelial cell specific protein 11 Hypothetical protein, full insert sequence [100% identity to Q6ZPL6, MKIAA1610 protein (Fragment) - Mus musculus (Mouse); 94.24% - Q8N161 Mesoderm induction early response 1 N2-β]

Proliferation-associated 2G4 [Mus musculus] Similar to SEPTIN6 type II

Endoplasmic reticulum protein ERp29 [precursor] Fatty acid-binding protein, epidermal (E-FABP) Histidine triad nucleotide-binding protein 1 (Adenosine 5′-monophosphoramidase) (Protein kinase C inhibitor 1) Programmed cell death 6 interacting protein (ALG-2 interacting protein X) (ALG-2 interacting protein 1) Prohibitin (B-cell receptor associated protein 32)

6.36 6.10 6.15 5.23

4.76

5.14

8.62

4.66

4.67 6.01

5.62 6.62 5.32 6.02 6.06 4.77

6.60

6.30

6.64

6.17

5.13

5.79

8.63

8.11 6.14

5.57

6.15

6.23 6.73 6.39

20565.58 52532.07 41592.55 37354.16

19462.17

14725.65

27070.34

23315.08

46325.88 23473.62

12258.94 127580.48 16605.01 38713.92 25206.62 31024.93

61540.39

61085.61

62195.75

46628.21

20855.43

10044.51

30624.31

37971.4 49680.57

29820.10

96010.20

28574.83 15059.38 13645.71

4.50

4.85 5.66

5.45 6.20 5.41 6.03 5.88 4.22

6.98, 7.36

6.73

6.30, 6.45

6.38

5.32

5.58-6.50 6.15-7.80 5.41

6.62 6.64

5.54

6.14, 6.25

6.09 6.20 6.25

24.8

50.0 23.4

9.8 48.5 15.6 35.3 20.9 29.8

61.5

59.5

60.0

44.7

18.5

24.0 30.3 8.0

44.6 48.2

27.0

99.0

25.9 14.5 13.1

25

11 16

9 23 8 19 12 12

30

19

23

13

8

11

14

20 16

15

30

15 15 19

90

100e 163e

60 71 116e 214e 66 79

167

132

92

1

1 1

1 1 1 1 1 1

2

1

2

1

1

72e 66

1

103

6

231e

1

386e 1 1

2

244e

104 207e

1 1 1

147e 90 148

5.05 6.13 6.12 5.55

5.00

4.96, 5.15 5.43

7.40-8.52

4.90 5.56 6.26 6.06 6.25 6.64 6.41

6.07, 6.16

5.72 6.45 6.24

16.8 52.2 42.1 14.7

22.7

13.2 12.8

27.8

20.3 28.0 45.8 46.5 49.8 49.5 24.0

96.0

21.4 13.0 13.0

8 21 22 10

11

16

18

9

14 15

14

22

15 19 11

3 3 1 1 1 1 1

226e 124e 83e 133 245e 65

1

1 3

2

2

1 1 1

219e

132e

82 99

174e

191e

156e 185e 82


research articles


784

Q8CEW4

P01946 Q60817 Q63024

Q8CEW4

Mus musculus ES cells cDNA, RIKEN full-length enriched library, clone:2410026K14 product:hypothetical protein, full insert sequence. [Fragment]

15197.34 23383.90 20734.72 Hypothetical protein 7.03 12475.19 HBA_RAT Q60817 Q63024

Q9D8U8 Q920C7


a Calculated for the corresponding SWISS-PROT entry using the ExPASy Compute pI/MW tool in ExPASy website (http://www.expasy.org). b Estimated from the position of the protein spot on the 2D gel. c Score is -10*Log (P), where P is the probability that the observed match is a random event (MASCOT, http://www.matrixscience.com/help/scoring-help.html). d Italic letters represent the accession numbers of NCBI database. e Data obtained with MALDI TOF/TOF.

1 87e 5.58

10.6

3

1 1 1 189e 107 62 8.06 4.12 7.56 7.93 4.52 5.41

11.7 35.7 14.4

10 9 11

1 1 107 93 20 15 47.8 30.2 6.26 5.69 SNX5_MOUSE Q920C7

no.

Sorting nexin 5 Carnitine deficiency-associated protein CDV3B (Pp36) Hemoglobin R-1 and R-2 chains R NAC/1.9.2. protein (R-NAC, nonmuscle form) Rat R-2u-globulin

MW (Da) pI


Table 1 (Continued)


theoreticala

pI

MW (kDa)

matches

scorec

no. of spot

pI

Yang et al.

Miscellaneous proteins 6.19 46797.36 5.84 29729.02

matches

observedb observedb

MW (kDa)

astrocytes neurons

scorec

no. of spot

research articles

Figure 4. Distribution of the identified proteins (A) and protein spots (B) from rat primary neurons (9) and astrocytes (0) in functional groups.

sequence and a MS/MS spectrum from m/z 1518.786 that assigned the identified sequence NICQFLIEIGLAK to eIF1 (Figure 5B). Identification of Low Molecular Weight Proteins. The fragmented form of P93 calcium-binding protein (Q9QV93) was not significantly identified with MS analysis because of the small size of the sequence (VTYKAPVPTG EVYFADSFDRGSL). After database search with PMF, a MS/MS spectrum from m/z 1770.836 was automatically acquired that assigned the identified sequence APVPTGEVYFADSFDR to the fragmented form of P93 calcium-binding protein (Figure 6). Combined MS and MS/MS database search provided unique and safe assignment with a significant Mascot score and 86% sequence coverage. In addition, other low molecular weight proteins, such as calmodulin (P62158; theoretical MW 16706.39 Da), stathmin (P13668; 17157.29 Da), eukaryotic translation initiation factor 1 (P48024; 12746.52 Da; Figure 5) and thioredoxin (P11232, 11542.28 Da) were unambiguously identified in neurons and astrocytes.

research articles

Proteomic Profiling of Rat Primary Neurons and Astrocytes Table 2. Superimposed Spots with Differently Identified Proteins in Figure 3 theoreticala no.

P47942 P63039 P21575 Q9WU78 P51635 P26516 P60891 Q9QWU4

O35331 O88989 P62994 O35244 P18422 Q63362


pI

MW (Da)

observedb pI

MW (kDa)

identityc matches

score

MS/MS

24 16 18 16

127 77 79 92

VPVGDQPPDIEFQIR FYNELTEILVR

Dihydropyrimidinase related protein-2 60 kDa heat shock protein Dynamin-1 Programmed cell death 6 interacting protein Alcohol dehydrogenase [NADP+] 26S proteasome non-ATPase regulatory subunit 7 Ribose-phosphate pyrophosphokinase I Glyceraldehyde-3-phosphate dehydrogenase

5.95 5.91 6.32 6.15

Neurons 62277.57 5.56 60955.49 95927.48 6.14 96010.20

6.81 6.29

36374.69 36539.82

6.90

36.0

14 13

119 136

DAGHPLYPFNDPY VVVHPLVLLSVVDHFNR

6.56

34703.04

7.10

32.3

14

102

VTAVIPCFPYAR

8.14

35827.99

13

95

Pyridoxal kinase Cytosolic malate dehydrogenase Growth factor receptor-bound protein 2 Peroxiredoxin 6 Proteasome subunit R type 3 NADH-ubiquinone oxidoreductase 13 kDa-B subunit

6.32 6.16 5.89 5.65 5.29 7.07

Astrocytes 34908.12 6.05 36483.11 25206.35 5.83 24687.40 28288.05 6.39 13280.59

14 5 18 11 12 8

85 51 152 86 66 65

57.1 99.0

34.2 24.1 11.2

(%)

11.54 12.10

9.66

11.04

LVINGKPITIFQER

11.36 FVEGLPINDFSR 10.14 14.78

a Calculated for the corresponding SWISS-PROT entry using the ExPASy Compute pI/MW tool in ExPASy website (http://www.expasy.org). b Observed pI and molecular weight refers to 2D-gel in Figure 3. c The alignment was performed using the ClustalW program (http://clustalw.genome.jp/) with deduced sequence from SWISS-PROT database.

Figure 5. MS-TOF spectrum of eukaryotic translation initiation factor 1 (A) and LIFT-TOF/TOF spectrum of the precursor ion detected at m/z 1518.786 (B). The identified sequence NICQFLIEIGLAK unambiguously assigned this protein spot to eukaryotic translation initiation factor 1 (P48024).

Discussion In the present study, we identified 226 and 228 different gene products in rat primary neurons and astrocytes using a proteomic approach based on MALDI TOF/TOF mass spectrometry. The major finding is that not only global and systematic protein expression data are shown in two major cell types of brainsneurons and astrocytessbut also a reliable protein chemical method is provided to identify cell type specific proteins independent of antibody availability and specificity. In addition, the high-throughput method provided concomitant identification of a large number of proteins including low molecular weight proteins, one of the limitations of MS

analysis. For example, a fragment form of P93 calcium-binding protein (theoretical MW: 2519.79 Da) was not identified with MS analysis but was unambiguously identified with MS/MS in our study (Figure 6). Most proteins were represented by a large number of multiple protein expression forms (MPEFs) and belong to the cytoskeleton. Tubulins (R and β forms) that play a role in neuronal polarity, were identified with 63 spots in neurons and 19 spots in astrocytes. It is known that most tubulin genes are expressed in nervous tissue and some appear to be enriched in or specific for neurons.16 On the other hand, actins, another major cytoskeleton class, were represented by 12 spots in Journal of Proteome Research • Vol. 4, No. 3, 2005 785

research articles

Yang et al.

Figure 6. MS-TOF spectrum of the fragmented form of P93 calcium-binding protein (A) and LIFT-TOF/TOF spectrum of m/z 1770.836 (B) that unambiguously assigned the sequence APVPTGEVYFADSFDR to P93 calcium-binding protein [Fragment] (Q9QV93).

neurons and 45 spots in astrocytes. A quite different number of MPEFs in given proteins may suggest different and typical composition of cytoskeleton proteins in neurons and astrocytes that are important for the differentiated function of these cell types. Cell type specific cytoskeleton proteins observed were as follows: neuronal tropomodulin, kinesin heavy chain isoform 5C and neuron-specific class III β-tubulin in neurons, and glial fibrillary acidic protein (GFAP) in astrocytes, a protein generally regarded as glial marker protein. The different expressional pattern of cytoskeleton proteins may reflect different backbones of cells making up morphology of neurons and astrocytes and that are also responsible for intracellular transport of materials and signaling.16,17 No major neuronal cytoskeleton element of the neurofilament class were identified in primary culture of neurons, confirming the previous finding of the absence of neurofilaments in HCN-2 neurons in culture.8 This observation may reflect the different physiological status as, e.g., differentiation and assembly of primary neurons in vitro. Classified proteins in Table 1 include 12 antioxidant proteins. Glutathione S-transferase P was identified in astrocytes but not in neurons. Glutathione S-transferase is known to be abundantly expressed in astrocytes unlike in neurons and take part in detoxification of toxic substances and xenobiotics including carcinogens, antibiotics and products of oxidative stress.18 From the aspect that astrocytes protect other brain cell types including neurons against active oxygen species,19 it may be possible that more antioxidant proteins were identified in astrocytes than neurons in this study. There is strong evidence that cellular mechanisms under heat shock response are differently regulated in various cell types in CNS.20,21 In this study, several chaperone proteins were identified and some showed cell-specific expression patterns. R B-Crystallin is expressed not only at high levels in astrocytes and oligodendrocytes but also in some neurons in the state of neurodegenerative disorders.20 We identified R B-Crystallin (P23928) and heat shock 27 kDa protein (P42930) in astrocytes. These proteins were suggested as significant proteins in astrocytes as to cause Alexander’s disease by stress reaction and were observed in Rosenthal fiber formation.22,23 786


The largest number of identified proteins belongs to metabolic pathways including glycolysis such as fructose-bisphosphate aldolase C (P09117), glyceraldehyde-3-phosphate dehydrogenase (Q9QWU4), phosphoglycerate kinase (P16617), phosphoglycerate mutase 1 (P25113) and R enolase (P04764) that were identified in both cells. γ enolase (P07323, neurons), generally regarded as neuronal marker protein, and fructosebisphosphate aldolase A (P05065, astrocytes) were also involved in glycolysis. Brain energy metabolism is highly compartmentalized, with certain metabolic pathways specifically localized in a given cell type. It is therefore not surprising that whereas ketone bodies can be oxidized by neurons, oligodendrocytes, and astrocytes, β-oxidation of free fatty acids is localized exclusively in astrocytes.24 We found δ 3,5-δ 2,4-dienoyl-CoA isomerase (Q62651), required for the oxidation of unsaturated fatty acids only in astrocytes. The pyruvate dehydrogenase complex catalyzes the overall conversion of pyruvate to acetyl-CoA and CO, and here two components of this complex were identified in neurons (dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (P08461), pyruvate dehydrogenase E1 component R subunit (P26284)) and pyruvate dehydrogenase E1 component β subunit (Q9D051) were expressed in both cells. Despite many studies about ammonia handling in brain, an important substrate and/or product of several enzymatic reactions in the brain, nitrogen signaling in CNS is still elusive. We identified several proteins involved in nitrogen metabolism in primary astrocytes. Glutamate dehydrogenase, predominantly located in astrocytes, catalyzes the oxidative deamination of glutamate to R-ketoglutarate, metabolic precursor for TCA, under physiological conditions.25 Lactate, alanine, citrate, and R-ketoglutarate, products of this reaction, are released by astrocytes and play a role in neuronal metabolism.26 On the other hand, glutamate is converted to glutamine by glutamine synthetase in the presence of ammonia. Nitrilase homologue 1(Q7TQ94) and nit protein 2 (Q9JHW2) involved in nitrogen synthesis were observed herein as well. Nit protein 2, a nitrogen regulatory protein, plays a role in expressional activation of many nitrogen catabolic genes27 and was previously reported

research articles


at the protein level in human brain from fetal Down syndrome with reduced expression.28 Mechanisms of translation initiation is a complex process in higher eukaryotes involving multiple isoforms of eukaryotic translation initiation factors (eIF) that work in concerted action and initiating tRNA, mRNA, 40 S ribosomal subunit, and 60 S ribosomal subunit to complete a translation initiation complex.29 We here describe for the first time astrocytic localization of eIF1 (P48024) using an analytical tool for unambiguous determination of this structure (Figure 5). eIF1 may play a role in ribosomal scanning and initiation codon selection.30 The identified eIF-4H (Q9WUK2) in primary astrocytes was predominantly localized in heart, liver and testis and at lower levels in brain, spleen, lung, skeletal muscle, kidney, and embryonic tissues (http://ca.expasy.org/cgi-bin/niceprot.pl?Q9WUK2). It probably takes part in translation initiation through proteinprotein interactions that possibly stabilize conformational changes that occur in eIF4A during RNA binding, ATP hydrolysis, and RNA duplex unwinding.29 These nucleic acid binding protein classes are a major target of current molecular biology studies but there is still limited information, and this proteomic approach significantly adds to knowledge in this area. The ubiquitin-proteasome pathway is involved in a variety of cellular activities such as cell cycle, signal transduction, apoptosis,31 metabolic regulation,32,33 and has been implicated in neuronal development and neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease.34 We identified 4 multiple expression forms of neuronal specific proteasomal protein, ubiquitin carboxyl-terminal hydrolase isozyme L1 (PGP9.5; Q9R0P9) in neurons only. PGP9.5 has been considered a neuronal marker protein35,36 and tumor marker for nonsmall-cell lung cancer.37 The finding suggests that the ubiquitin-proteasome system may be different between neurons and astrocytes such as tissue specific distribution of proteasomes, immunoproteasomes, and their regulators.32 The composition of proteasomal subunits may be modulated based upon specific roles of neurons and astrocytes in the CNS.36 The signaling protein 14-3-3 is widely distributed in CNS and is expressed most abundantly in neurons.38 14-3-3 protein ζ/δ was identified in primary cultured neurons in this study while the other isoforms, 14-3-3D and E were reported in the conditioned medium of cultured mouse astrocytes as secreted proteins.39 14-3-3 protein ζ/δ may play critical roles in cell signaling events by Ca2+/calmodulin-dependent protein kinase II and protein kinase C that control progress through the cell cycle, transcriptional alterations and programmed cell death.40 At this point a statement on the use of protein profiling to search cell-specific proteins has to be delivered. Among signaling proteins, several isoforms of annexins (I, II, III, IV, and VII) were exclusively identified in astrocytes in this study. However, annexin I, II, IV, and V were reported in human cortical neuronal cell line HCN-28 and annexin II, V, VI, and VII in primary cortical neurons by LC-MS/MS.10 However specific proteomic methods are, low abundance proteins i.e., proteins at low concentrations cannot be detected and this may be the case when we observed absence of a protein. Some proteins shown in astrocytes examined in this study have been reported in neurons: Staufen (Q6TNG5), a component of mRNA transport, was reported at the mRNA level in neurons41 and phosphatidylinositol transfer protein R isoform (P16446) expression was detected in almost all neurons throughout the entire adult brain at the transcriptional level by in situ hybridization histochemistry.42 On the other hand, β-synuclein

(Q91ZZ3) was identified in neurons in this study whereas expression was also reported in normal human astrocytes.43 Due to space limitation discussion of all differentially expressed proteins is not possible and may not be necessary as the absence or presence of a given protein is self-explaining. The level set for identification was detectable or nondetectable and only high-abundance proteins (i.e., Coomassie blue stained spots) were identified by this approach. Cell culture conditions responsible for differential expression of proteins have to be taken into account as well but we intended to show neurons and astrocytes in the media generally used in neuroscience protocols to avoid metabolic derangement. Cell cycle differences have to be respected as well as potential confounding factors. Splicing variants and posttranslational modifications may be represented by the multiple expression forms and these were not studied so far. We are aware of the possible artifacts and inherent problems of studying proteomes but analysis of proteins herein is fair and unambiguous. We also show a confounding factor that has to be indicated to the innocent reader: As shown in Table 2 proteins of superimposed spots can be identified but of course not quantified.

Conclusions We constructed a 2D reference database for primary cultured neurons and astrocytes from rat cerebellum with 428 and 406 protein spots representing 226 and 228 individual proteins, respectively, using an analytical tool independent of antibody availability and specificity. Proteins identified belong to several protein classes and cell specific patterns were observed. Several potentially specific neuronal- or astrocytic- candidate marker proteins such as, e.g., neuronal tropomodulin, kinesin heavy chain isoform 5C, neuron-specific class III β-tubulin, ubiquitin carboxyl-terminal hydrolase isozyme L1 in neurons, and glial fibrillary acidic protein and eukaryotic translation initiation factor 1 in astrocytes are proposed. This comparative proteomics study forms the basis for future work on neuron and astrocyte protein expression and is a first valuable guide for designing comparative studies at the protein level.

Acknowledgment. This study was generously supported by the Red Bull company, Salzburg, Austria. References (1) Langen, H.; Berndt, P.; Roder, D.; Cairns, N.; Lubec, G.; Fountoulakis, M. Two-dimensional map of human brain proteins. Electrophoresis 1999, 20, 907-916. (2) Fountoulakis, M.; Juranville, J. F.; Dierssen, M.; Lubec, G. Proteomic analysis of the fetal brain. Proteomics 2002, 2, 1547-1576. (3) Yang, J. W.; Czech, T.; Lubec, G. Proteomic profiling of human hippocampus. Electrophoresis 2004, 25, 1169-1174. (4) Lubec, G.; Krapfenbauer, K.; Fountoulakis, M. Proteomics in brain research: potentials and limitations. Prog. Neurobiol. 2003, 69, 193-211. (5) Gauss, C.; Kalkum, M.; Lowe, M.; Lehrach, H.; Klose, J. Analysis of the mouse proteome. (I) Brain proteins: separation by twodimensional electrophoresis and identification by mass spectrometry and genetic variation. Electrophoresis 1999, 20, 575600. (6) Fountoulakis M.; Schuller, E.; Hardmeier, R.; Berndt, P.; Lubec, G. Rat brain proteins: two-dimensional protein database and variations in the expression level. Electrophoresis 1999, 20, 35723579. (7) Beranova-Giorgianni, S.; Pabst, M. J.; Russell, T. M.; Giorgianni, F.; Goldowitz, D.; Desiderio, D. M. Preliminary analysis of the mouse cerebellum proteome. Brain. Res. Mol. Brain. Res. 2002, 98, 135-140.


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PR049774V

Proteome Analysis of Primary Neurons and Astrocytes from Rat

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