Population characteristics
The baseline characteristics of dogs in proteomic study are shown in Table 1. There were no significant differences in sex, weight, and breed among the three groups. The haematological and blood chemistry which evaluated kidney and liver function were not significantly different between the three groups. The protein in plasma was also not significantly different between the three groups and within each group.
Table 1 The baseline characteristics of dogs include in proteomic study
Group
|
No
|
Age (years)
|
Sex
|
Weight (Kg)
|
Breed
|
WBC (x103)
|
Hct (%)
|
ALT
(U/L)
|
Creatinine (mg/dL)
|
Protein (μg/µl)
|
CCDS
|
1
|
15
|
F
|
17
|
Mongrel
|
5.3
|
38
|
80
|
1.13
|
3.53
|
2
|
13
|
F
|
11
|
Mongrel
|
16
|
41.9
|
195
|
0.84
|
3.57
|
3
|
17
|
M
|
23
|
Mongrel
|
10.8
|
33.6
|
55
|
0.94
|
3.42
|
4
|
12
|
F
|
20
|
Mongrel
|
12.1
|
39.2
|
95
|
1.33
|
3.55
|
Ageing
|
1
|
15
|
F
|
15
|
Mongrel
|
13.4
|
38
|
77
|
1.77
|
3.89
|
2
|
12
|
M
|
25
|
Mongrel
|
6.9
|
39.4
|
179
|
1.52
|
3.92
|
3
|
12
|
F
|
19
|
Mongrel
|
10.5
|
36
|
304
|
1.65
|
3.87
|
4
|
12
|
F
|
30
|
Mongrel
|
10.2
|
43
|
236
|
1.23
|
3.92
|
Adult
|
1
|
3
|
M
|
14
|
Mongrel
|
14.2
|
36.5
|
22
|
1.25
|
4.04
|
2
|
1
|
M
|
14
|
Mongrel
|
6.9
|
40
|
175
|
1.55
|
3.84
|
3
|
3
|
M
|
13
|
Mongrel
|
13.7
|
34
|
26
|
1.6
|
4.01
|
4
|
1
|
F
|
13
|
Mongrel
|
6.3
|
39
|
76
|
1.35
|
3.74
|
White blood cells (WBC); p = 0.9036, Haematocrit (Hct); p = 0.8126, Alanine aminotransferase (ALT); p = 0.0976, creatinine; p = 0.0548
Correlation between the CCDR score and plasma Aβ42 levels
First, levels of plasma Aβ42 in CCDS dogs (n=10) were lower than those in ageing dogs (n=7) but higher than those in the adult group (n=4) (for Aβ42± SD: 75.40 ± 101 pg/mL in CCDS, 179.21 ± 185.6 pg/mL in the ageing group and 5.88 ± 9.28 pg/mL in the adult group). The Aβ42 level in the ageing and adult group was shown a significant difference (* p = 0.038) Nevertheless, there were no significant differences between CCDS with other groups. (Fig. 1a). Furthermore, clinical diagnosis of the CCDS group was performed with the CCDR questionnaire. CCDR scores above 50 are indicative of CCDS in older companion dogs. Second, we evaluated individual plasma Aβ42 levels in all dogs. Our study showed that plasma Aβ42 levels in the CCDS group were not correlated with the CCDR score
(p=0.131, R2=0.261) (Fig. 1b).
Fig. 1 Plasma Aβ42 concentration (a). Plasma Aβ42 concentrations (pg/μL) in each group (*p = 0.038) (b). Plasma Aβ42 concentrations and CCDR scores in the CCDS group
Proteomics profile
Plasma proteins of the CCDS group were compared with others by a proteomics approach to study differential protein expression. The proteins were separated by One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Each lane was cut into 11 pieces (Fig. 2a.) prior to in-gel trypsin digestion. The digested peptides were identified by nano-LC-MS/MS analysis, and each protein was quantified using an exponentially modified protein abundance index (emPAI) value from the label-free spectral counting technique. In total, 1037 proteins were identified in the plasma samples from domestic dogs in Thailand against a non-redundant National Center for Biotechnology Information (NCBInr) database specific to Mammalia spp. as a taxonomic filter.
The protein bands in SDS-PAGE of CCDS samples in rows 5, 7 and 10 were different than those of the other groups by macroscopic appearance (Fig. 2a). Data from nano-LC-MS/MS unveiled proteins in band 5 of CCDS samples that were composed of immunoglobulin gamma heavy chain B, fibrinogen beta chain, alpha-2-HS-glycoprotein, fibrinogen gamma chain, immunoglobulin gamma heavy chain C, vitamin D-binding protein, beta-2-glycoprotein 1 precursor and immunoglobulin A heavy chain constant region. The proteins in band 7 of CCDS samples were composed of haptoglobin, complement C4-A and immunoglobulin lambda-like polypeptide 5-like. The proteins in band 10 of CCDS samples were composed of haptoglobin.
Fig. 2 Protein patterns using (a). SDS-PAGE with Coomassie blue R-250 staining (original gel image was in supplement information) (b). Heat map of differentially expressed protein patterns
Heat map analysis, presenting differential capability of protein expression, illustrated 3 distinct groups (Fig. 2b). For species specificity, proteomic data were identified only for Canis spp. A Venn diagram analysis shows the number of proteins overlapping between the three datasets. A total of 87 canine proteins were matched, and 35 (40.2%) of those were detected in all three groups. The number of proteins identified overlapping between CCDS and ageing group was 48 (55.1%) and number of proteins identified overlapping between CCDS and adult group was 41 (47.1%) (Fig. 3).
Fig. 3 Venn diagram of protein detection (Canis spp.)
The most upregulated proteins in the CCDS group compared with the adult group were involved with the coagulation cascade, while the most upregulated proteins in the CCDS group compared with the ageing group were involved with apolipoprotein. The most downregulated proteins in the CCDS group compared with the adult group or the ageing group were alpha-2-macroglobulin and alpha-1B-glycoprotein. Quantification was performed using emPAI provided by Mascot. The emPAI values in this report were the mean of three biological replications. A different expression of proteins at least two replicates were reported as altered proteins. In Fig. 4, the top ten most upregulated and downregulated proteins in comparisons between the CCDS group and the adult group are demonstrated in (a) and (b), respectively. Whereas, the top ten most upregulated and downregulated proteins in comparisons between the CCDS group and the ageing group are presented in (c) and (d), respectively.
Fig. 4 The changes in most proteins ranked by emPAI value: (a). Upregulated proteins in comparisons between the CCDS group and the adult group (b). Downregulated proteins in comparisons between the CCDS group and the adult group (c). Upregulated proteins in comparisons between the CCDS group and the ageing group (d). Downregulated proteins in comparisons between the CCDS group and the ageing group
Gene Ontology (GO) and pathway enrichment analyses of proteins
We first mapped the proteins onto GO databases via the PANTHER database using 3 primary categories: molecular function and biological process. The gene ontology analysis provided the overview of functional interpretation of the resultant differential protein expression. In this study, three general terms were used to classified the differential dataset as biological process, molecular function, and cellular component. In the GO molecular function category, the upregulated proteins in comparisons between the CCDS group and both the ageing group and the adult group were similarly classified into 4 groups: binding, molecular function regulator, catalytic activity and transporter activity. In the GO biological process category, the upregulated proteins in comparisons between the CCDS group and both the ageing group and the adult group were similarly classified into 4 groups: metabolic process, cellular process, biological regulation and transporter activity (Fig. 5).
Pathway enrichment analysis of CCDS proteins of interest using STRING showed some relation between amyloid precursor protein and some proteins of interest. Proteins at the core of the traffic link have good protein-protein interactions.
Fig. 5 Gene Ontology molecular function and biological process categories for upregulated proteins in the CCDS group: (a). comparison of the CCDS group with the ageing group in molecular function; (b). comparison of the CCDS group with the adult group in molecular function; (c). comparison of the CCDS group compared with the ageing group in biological process; and (d). comparison of the CCDS group with the adult group in biological process
From 87 of proteins that matched, among which 48 and 41 proteins showed at least 1.5-fold differences in their expression levels according to the emPAI values in the CCDS vs ageing and CCDS vs adult group in comparison. The downregulated proteins in comparisons of the CCDS group with both the adult group and the ageing group included 4 proteins: alpha-2-macroglobulin, alpha-1B-glycoprotein, complement factor B and immunoglobulin lambda-like polypeptide 5-like. The downregulated proteins, involved in blood coagulation and the complement cascade. The upregulated proteins in the comparisons of the CCDS group with both the ageing group and the adult group were specifically involved in several biological processes. The biological process of upregulated proteins linked to neurodegenerative disease was mostly blood coagulation, acute phase protein and complement cascade, as shown in Table 2.
Table 2. Downregulated and upregulated proteins in comparisons of the CCDS group with both the adult and the ageing
Downregulated proteins
|
Accession numbera
|
Protein name
|
Protein mass
|
pI
|
Protein score
|
Biological process
|
P-value
|
gi|345792424
|
alpha-2-macroglobulin
|
165114
|
6.27
|
101
|
negative regulation of complement activation
|
p = 0.0144
|
gi|545487024
|
alpha-1B-glycoprotein
|
61261
|
5.81
|
47
|
platelet degranulation
|
p = 0.0001
|
gi|345778397
|
complement factor B
|
86266
|
7.18
|
75
|
regulation of complement activation
|
p = 0.1544
|
gi|545544683
|
immunoglobulin lambda-like
polypeptide 5-like
|
24739
|
6.41
|
1116
|
innate immune response
|
p = 0.0049
|
Upregulated proteins
|
Accession numbera
|
Protein name
|
Protein mass
|
pI
|
Protein score
|
Biological process
|
P-value
|
gi|73955106
|
apolipoprotein A-I
|
30163
|
5.28
|
2244
|
lipoprotein metabolic process
|
p = 0.0136
|
gi|704000372
|
apolipoprotein A-IV
|
42510
|
5.75
|
318
|
removal of superoxide radicals
|
p < 0.0001
|
gi|345799905
|
predict apolipoprotein A-IV
|
43795
|
5.34
|
615
|
removal of superoxide radicals
|
p < 0.0001
|
gi|545488191
|
apolipoprotein E isoform X5
|
47029
|
8.45
|
88
|
regulation of amyloid beta clearance
|
N/A
|
gi|73978329
|
fibrinogen alpha chain
|
96583
|
5.76
|
275
|
blood coagulation
|
p = 0.002
|
gi|73977992
|
fibrinogen gamma chain isoformX1
|
49286
|
5.74
|
1092
|
blood coagulation
|
p < 0.0001
|
gi|120141
|
fibrinogen gamma chain, partial
|
2688
|
4.55
|
93
|
blood coagulation
|
p = 0.20
|
Accession numbera
|
Protein name
|
Protein mass
|
pI
|
Protein score
|
Biological process
|
P-value
|
gi|57109938
|
kininogen-1
|
48317
|
5.58
|
104
|
blood coagulation
|
p < 0.0001
|
gi|545485785
|
plasminogen isoformX1
|
90952
|
6.75
|
121
|
blood coagulation
|
p = 0.1496
|
gi|130314
|
plasminogen
|
36654
|
8.48
|
152
|
blood coagulation
|
p < 0.0001
|
gi|123511
|
haptoglobin
|
36434
|
5.72
|
2272
|
acute phase response
|
p = 0.0001
|
gi|545560457
|
inter-alpha-trypsin inhibitor heavy
chain H4 isoformX1
|
113355
|
7.1
|
292
|
acute phase response
|
p < 0.0001
|
gi|359321961
|
prothrombin
|
70259
|
5.71
|
42
|
acute phase response and blood coagulation
|
N/A
|
gi|345803075
|
C4b-binding protein alpha chain isoform X1
|
68505
|
7.77
|
171
|
complement activation classical pathway
|
p < 0.0001
|
gi|50979240
|
clusterin precursor
|
51757
|
5.65
|
107
|
complement activation and regulation of Aβ formation
|
p < 0.0001
|
gi|598107
|
IgA heavy chain constant region
|
37255
|
6.06
|
114
|
complement activation classical pathway
|
p < 0.0001
|
gi|19715661
|
Ig J chain
|
12733
|
4.94
|
38
|
innate immune response
|
p = 0.0789
|
gi|73995687
|
Ig lambda-like polypeptide 5-like
|
14832
|
8.84
|
1528
|
complement activation classical pathway
|
p < 0.0001
|
gi|345777714
|
alpha-1-acid glycoprotein 1 isoform X1
|
23291
|
5.38
|
45
|
regulation of immune response
|
p = 0.0245
|
gi|545531456
|
plasma protease C1 inhibitor
|
48128
|
5.51
|
88
|
complement activation classical pathway
|
p < 0.0001
|
Accession numbera
|
Protein name
|
Protein mass
|
pI
|
Protein score
|
Biological process
|
P-value
|
gi|50978658
|
alpha-fetoprotein precursor
|
68738
|
5.77
|
52
|
cellular protein metabolic process
|
p = 0.027
|
gi|256574824
|
glutathione peroxidase 3 precursor
|
25363
|
8.79
|
59
|
response to oxidative stress
|
p < 0.0001
|
gi|44888810
|
hemoglobin alpha chain
|
15208
|
7.98
|
267
|
cellular oxidant detoxification
|
p < 0.0001
|
gi|73988725
|
hemopexin
|
51305
|
6.88
|
149
|
heme metabolic process
|
p < 0.0001
|
gi|119637837
|
pigment epithelium-derived factor
|
44236
|
8.69
|
40
|
aging
|
p < 0.0001
|
gi|57089193
|
transthyretin isoform 2
|
15858
|
6.42
|
619
|
retinol, thyroid hormone transport
|
p = 0.001
|
a Accession number from NCBInr database for Canis spp.
N/A = cannot measure by ANOVA because the samples all have a standard error of zero
The downregulated and upregulated proteins which reported in the table 2 showed protein score higher than 95% in confidence. The differential expression was shown from the semi-quantification by selecting the altered proteins with at least two replicates. To explore the potential proteins, we performed a pathway analysis by using STRING version 11.0. Total protein changes in comparisons of the CCDS group with both the ageing group and the adult group were expanded to show the evidence of an interaction, giving a total of 24 proteins. We compared this protein set to those of the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathways databases. Most proteins involved with the Gene Ontology biological process category are stress response proteins. Most proteins involved with the KEGG and Reactome pathway databases are complement and coagulation cascade and immune system proteins, respectively (Fig. 6).
Fig. 6 STRINGS protein-protein interaction: Analysis of protein changes in the CCDS group compared with both the adult group and the ageing group (total proteins = 24, red colour = response to stress, blue colour = complement and coagulation cascades and green = innate immune system)