3.4. Discussion
5.3.3. Heatmap visualisation
Protein quantification using heat map visualisation was based on the detected peak areas of the top three unique peptide ratios calculated from the raw MS/MS data. The peak area is illustrated in a spectrum from red to black to green. Average peak areas are shown in black, whilst smaller peak areas are shown in green and larger peak areas are shown in red. The gradation of the change in peak areas is indicated by the intensity of the green or red colour.
111
Rank Protein P-value % change
1 Glutathione S-transferase Mu 5 (Fragment) 1.65×10-4 -17%
2 Glutamine synthetase 9.64×10-4 -16%
3 Vimentin 9.68×10-4 100%
4 Angiotensin 1 converting enzyme (Peptidyl-dipeptidase(A) 1 2.02×10-3 62%
5 Phospholemman 2.42×10-3 31%
6 Reticulon-4 (RTN4) 2.83×10-3 -25%
7 Nucleoside diphosphate kinase A 4.52×10-3 14%
8 DEAH (Asp-Glu-Ala-His) box polypeptide 15, isoform CRA_a 4.68×10-3 28% 9 Protein Spnb1 (Beta-I spectrin) 5.60×10-3 -15% 10 Glial fibrillary acidic protein 8.80×10-3 105% 11 Delta-1-pyrroline-5-carboxylate dehydrogenase, mitochondrial 9.72×10-3 27%
12 Rbp1 protein 1.34×10-2 37%
13 Heat shock 70kD protein 5 (Glucose-regulated protein) 1.35×10-2 14% 14 Sodium- and chloride-dependent GABA transporter 1 1.43×10-2 -12% 15 Gamma-aminobutyric acid receptor subunit beta-1 1.55×10-2 -21% 16 Keratin, type II cytoskeletal 5 1.76×10-2 -66% 17 Dehydrogenase/reductase SDR family member 1 2.41×10-2 43%
18 Podoplanin 2.47×10-2 77%
19 Calponin-3 2.50×10-2 53%
20 Heat shock protein HSP 90-beta 2.54×10-2 14%
21 Barrier-to-autointegration factor 2.54×10-2 16% 22 S100 calcium binding protein A13 2.58×10-2 29% 23 CaM kinase-like vesicle-associated protein 2.61×10-2 -12.43 24 Eukaryotic translation initiation factor 2, subunit 3, structural
gene X-linked 2.74×10
-2 10%
25 Peripheral plasma membrane protein CASK 2.76×10-2 -10%
26 Ribonuclease inhibitor 2.81×10-2 28%
27 S100 protein, beta polypeptide, neural, isoform CRA_b 2.83×10-2 36% 28 Uncharacterised protein (Fragment) F6QH50 2.85×10-2 -20% 29 Complement C1q subcomponent subunit B 2.86×10-2 38%
30 Moesin 2.87×10-2 52%
31 Uncharacterised protein (Fragment) F8WHU8 2.87×10-2 17% 32 Probable cationic amino acid transporter 3.00×10-2 11%
33 Hspb1 protein 3.16×10-2 140%
34 Short coiled-coil protein 3.36×10-2 40%
35 Small nuclear ribonucleoprotein Sm D3 3.38×10-2 7% 36 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit
7 3.70×10
-2 -7%
37 Clusterin 3.76×10-2 40%
38 Potassium inwardly-rectifying channel J3 3.91×10-2 -16%
Table 5.5. Proteins (n=38) whose expression was significantly changed in the hippocampus of C57BL/6J mice 7 days after administration of kainic acid followed by PSD95BP, when compared to hippocampi of mice that received PSD95BP alone (n=4 per group). Homogenised hippocampal tissues were analysed using liquid chromatography mass spectrometry, and proteins identified based on the peptide sequences detected and queried through Mascot. Results are ranked in the order of p-value (lowest to highest), with percentage change in expression indicated (a negative percentage change represents a down-regulation). Statistical significance (p<0.05) was determined by one-way Analysis of Variance with Bonferroni correction for multiple comparisons.
112
Rank Protein P-value % change
1 Cystatin C 2.33×10-5 28%
2 Moesin 1.82×10-3 91%
3 Rbp1 protein 1.98×10-3 41%
4 Glutamine synthetase 2.04×10-3 -15%
5 BolA-like protein 1 2.13×10-3 -18%
6 Corticosteroid 11-beta-dehydrogenase isozyme 1 (Fragment) 2.51×10-3 -66% 7 Glial fibrillary acidic protein 2.70×10-3 116% 8 Complement C1q subcomponent subunit C 4.68×10-3 46% 9 Peroxisomal acyl-coenzyme A oxidase 1 4.76×10-3 66% 10 Complement C1q subcomponent subunit B 5.08×10-3 42% 11 NADH dehydrogenase [ubiquinone] flavoprotein 1,
mitochondrial 5.52×10
-3 10%
12 Cytosolic 10-formyltetrahydrofolate dehydrogenase 6.20×10-3 26%
13 Alanine aminotransferase 1 6.88×10-3 -32%
14 Uncharacterised protein putative 8.44×10-3 -16% 15 Heterogeneous nuclear ribonucleoprotein A1 8.76×10-3 17% 16 Glycoprotein m6a, isoform CRA_b 9.52×10-3 -13%
17 Vimentin 1.02×10-2 114%
18 Peroxisomal multifunctional enzyme type 2 1.06×10-2 20%
19 Galectin-1 1.16×10-2 77%
20 Phytanoyl-CoA hydroxylase-interacting protein 1.24×10-2 -17%
21 CD44 antigen 1.28×10-2 211%
22 Ras-related protein Rab-8B 1.30×10-2 -15%
23 Uncharacterised protein E9PWP6 1.50×10-2 -212%
24 Ribosomal protein 1.56×10-2 6%
25 Acetyl-CoA acetyltransferase, cytosolic 1.71×10-2 30% 26 UPF0160 protein MYG1, mitochondrial (Fragment) 1.77×10-2 173% 27 CaM kinase-like vesicle-associated protein 1.93×10-2 -17%
28 Calmodulin 1.95×10-2 -9%
29 Uncharacterised -protein Yjefn3 (Fragment) 2.16×10-2 -51% 30 Microtubule-associated serine/threonine-protein kinase 3 2.18×10-2 -13%
31 Catalase 2.24×10-2 24%
32 Cannabinoid receptor 1 (Brain) 2.30×10-2 -30% 33 S100 calcium binding protein A13 2.34×10-2 19%
34 Dgkh protein 2.35×10-2 -32%
35 Alpha globin 1 2.76×10-2 33%
36 G protein-regulated inducer of neurite outgrowth 1 3.17×10-2 -11% 37 Acidic leucine-rich nuclear phosphoprotein 32 family member B 3.51×10-2 47%
38 RTN4 3.59×10-2 -27%
39 Tial1 cytotoxic granule-associated RNA binding protein-like 1,
isoform CRA_c 3.60×10
-2 13%
40 Neuroligin-2 3.64×10-2 -22%
41 Kalirin 3.86×10-2 -44%
Table 5.6. Proteins (n=41) whose expression was significantly changed in the hippocampus of C57BL/6J mice 7 days after administration of kainic acid followed by 1400W, when compared to hippocampi of mice that received 1400W alone (n=4 per group). Homogenised hippocampal tissues were analysed using liquid chromatography mass spectrometry, and proteins identified based on the peptide sequences detected and queried through Mascot. Results are ranked in the order of p-value (lowest to highest), with percentage change in expression indicated (a negative percentage change represents a down-regulation). Statistical significance (p<0.05) was determined by one-way Analysis of Variance with Bonferroni correction for multiple comparisons.
113
Rank Protein P-value % change
1 Phosphatidate cytidylyltransferase 1 1.70×10-3 -38%
2 Gene model 1604B, (NCBI) 2.66×10-3 235%
3 Basigin 3.73×10-3 -18%
4 Angiotensin 1 converting enzyme (Peptidyl-dipeptidase(A) 1 4.36×10-3 146% 5 Putative ATP-dependent Clp protease proteolytic subunit,
mitochondrial 4.88×10
-3 17%
6 Glycylpeptide N-tetradecanoyltransferase 7.12×10-3 -15% 7 Synaptic vesicle membrane protein VAT-1 homolog-like 8.32×10-3 94%
8 Nitric oxide synthase, brain 8.92×10-3 43%
9 Potassium inwardly-rectifying channel J3 9.76×10-3 -22% 10 Mammalian ependymin-related protein 1 1.42×10-2 22% 11 Proline-rich AKT1 substrate 1 (Fragment) 1.49×10-2 -18%
12 Sideroflexin-5 1.50×10-2 -12%
13 CAP-Gly domain-containing linker protein 2 1.56×10-2 -47%
14 Ataxin-2-like protein 1.64×10-2 -37%
15 Proprotein convertase subtilisin/kexin type 1 inhibitor 1.64×10-2 44%
16 Alpha-adducin 1.68×10-2 -24%
17 Protein kinase C beta type 1.80×10-2 -17%
18 Calbindin-28K 1.84×10-2 52%
19 Integral membrane protein 2B 2.07×10-2 -21%
20 Acyl-CoA thioesterase 11 2.10×10-2 -26%
21 Small nuclear ribonucleoprotein-associated protein 2.30×10-2 -19% 22 BTB/POZ domain-containing protein 17 2.62×10-2 -18% 23 Putative uncharacterised protein 2.63×10-2 -20%
24 Somatostatin 2.66×10-2 72%
25 Protein Dos 2.67×10-2 50%
26 Cytochrome b-c1 complex subunit 10 2.96×10-2 -17% 27 Neural visinin-like type 1 protein 3.00×10-2 -12% 28 Long-chain-fatty-acid--CoA ligase 3 3.08×10-2 -13%
29 Copine-2 3.28×10-2 119%
30 Plasma membrane calcium-transporting ATPase 2 3.59×10-2 -14% 31 Nuclear protein localisation protein 4 homolog 3.78×10-2 38%
Table 5.7. Proteins (n=31) whose expression was significantly changed in the hippocampus of C57BL/6J mice 7 days after administration of kainic acid followed by PSD95BP, when compared to hippocampi of mice that received KA alone (n=4 per group). Homogenised hippocampal tissues were analysed using liquid chromatography mass spectrometry, and proteins identified based on the peptide sequences detected and queried through Mascot. Results are ranked in the order of p-value (lowest to highest), with percentage change in expression indicated (a negative percentage change represents a down-regulation). Statistical significance (p<0.05) was determined by one-way Analysis of Variance with Bonferroni correction for multiple comparisons.
114
Rank Protein P-value % change
1 Src substrate cortactin 1.94×10-5 -12%
2 Sideroflexin-1 2.33×10-3 15%
3 Acid ceramidase 2.99×10-3 63%
4 Ataxin-10 5.20×10-3 39%
5 Basigin 6.32×10-3 -17%
6 CAP-Gly domain containing linker protein 2 6.60×10-3 -47%
7 Albumin 1 6.68×10-3 69%
8 Prosaposin, isoform CRA_e 7.72×10-3 79%
9 Ferritin heavy chain 9.40×10-3 17%
10 G protein-regulated inducer of neurite outgrowth 1 1.26×10-2 -14%
11 Myosin-11 1.35×10-2 85%
12 Tubulointerstitial nephritis antigen-like 1 1.39×10-2 29% 13 Quinone oxidoreductase-like protein 2 1.46×10-2 90% 14 Protein kinase, cAMP dependent regulatory, type II alpha 1.49×10-2 16%
15 MCG5603 1.72×10-2 -31%
16 Protein Gm8994 1.72×10-2 33%
17 Peroxisomal acyl-coenzyme A oxidase 1 1.82×10-2 24% 18 Uncharacterised protein (putative) 1.96×10-2 -21%
19 Protein SOGA3 1.98×10-2 -11%
20 D-dopachrome tautomerase 1.99×10-2 -33%
21 Signal peptidase complex subunit 2 2.06×10-2 -30% 22 NADH-ubiquinone oxidoreductase chain 1 2.18×10-2 33% 23 Acetyl-CoA acetyltransferase, cytosolic 2.33×10-2 29% 24 Glutamate [NMDA] receptor subunit epsilon-1 2.56×10-2 -23$%
25 Integral membrane protein 2B 2.59×10-2 -13%
26 Folh1 protein 2.70×10-2 23%
27 Echinoderm microtubule-associated protein-like 2 2.81×10-2 36%
28 MCG5232 3.00×10-2 83%
29 Immunity-related GTPase family Q protein 3.14×10-2 -27% 30 Presequence protease, mitochondrial 3.14×10-2 18% 31 Synaptic vesicle membrane protein VAT-1 homolog-like 3.21×10-2 95%
32 Metallothionein 3.29×10-2 -16%
33 Phospholipase D3 3.36×10-2 -18%
34 Small ubiquitin-related modifier 2 3.44×10-2 -14%
35 Alpha globin 1 3.72×10-2 63%
36 Protein Pzp 3.88×10-2 123%
Table 5.8. Proteins (n=36) whose expression was significantly changed in the hippocampus of C57BL/6J mice 7 days after administration of kainic acid followed by 1400W, when compared to hippocampi of mice that received KA alone (n=4 per group). Homogenised hippocampal tissues were analysed using liquid chromatography mass spectrometry, and proteins identified based on the peptide sequences detected and queried through Mascot. Results are ranked in the order of p-value (lowest to highest), with percentage change in expression indicated (a negative percentage change represents a down-regulation). Statistical significance (p<0.05) was determined by one-way Analysis of Variance with Bonferroni correction for multiple comparisons.
115 In the PSD95BP analysis, a total of 20 proteins were shortlisted using heat map visualisation. The peak areas of the majority of these proteins were increased following KA-induced seizures, whereas those that received vehicle rather than KA had peak areas that were typically lower than average (Figure 5.1). Administration of PSD95BP led to an increase in peak area for a number of proteins, in comparison to controls that received vehicle alone (Figure 5.1). When PSD95BP was given to animals that had previously experienced KA-induced seizures, the increase in peak areas was compounded and was consistent with an additive effect of the two compounds given alone (Figure 5.1).
In the 1400W analysis, a total of 22 proteins were shortlisted using heat map visualisation. As in the PSD95BP analysis, KA administration increased peak areas of the majority of proteins, while the corresponding vehicle treatment had the opposite effect (Figure 5.2). Likewise, treatment with 1400W increased the peak areas of several proteins when compared to vehicle-only controls and had an additive effective, in terms of numbers of proteins with increased peak area, when given to animals that had previously received KA (Figure 5.2).