Profiling the alterations of serum proteome in dairy cows with retained placenta using high-throughput tandem mass tags quantitative approach

Figures & data

Figure 1. Statistical analysis of proteomics results: A) Volcano plot showing the differences in the abundance of proteins between the retained placenta (RP; n = 9) and the physiological puerperium (PP; n = 6) groups; B) Principal Component Analysis (PCA) score plot showing samples from cows with RP and PP.

Table 1. The proteins with the different relative abundance (ratio between the amount in an individual sample and internal standard) in the cows with the retained placenta (RP; n = 9) and those with the physiological puerperium (PP; n = 6).

			Abundance
			median (min − max)
Accession	Gene	Description	RP (n = 9)	PP (n = 6)	FC	P − value	FDR
Higher abundance in the RP group
Q2TBU0	HP	Haptoglobin	1.10 (0.95 − 1.67)	0.59 (0.49 − 0.80)	1.86	<0.001	0.010
A0A4W2I9H0	GPX3	Glutathione peroxidase	1.22 (0.92 − 1.51)	0.86 (0.75 − 0.96)	1.42	0.002	0.026
A0A4W2HPU3	BPI	Lipopolysaccharide-binding protein	1.20 (1.05 − 1.48)	0.85 (0.75 − 0.89)	1.41	<0.001	0.010
Q3T052	ITIH4	Inter-alpha-trypsin inhibitor heavy chain H4	1.07 (0.93 − 1.39)	0.79 (0.55 − 0.97)	1.35	0.002	0.023
A0A3Q1M032	IGHM	Uncharacterized protein*	1.01 (0.93 − 1.49)	0.81 (0.74 − 0.88)	1.25	<0.001	0.009
A0A4W2H221	FGA	Fibrinogen alpha chain	1.08 (0.91 − 1.51)	0.88 (0.84 − 1.38)	1.22	0.003	0.042
A0A4W2EKG5	HPX	Hemopexin	1.10 (1.01 − 1.20)	0.92 (0.86 − 0.97)	1.20	0.004	0.010
G5E5T5	IGHM	Uncharacterized protein*	1.01 (0.86 − 1.49)	0.84 (0.79 − 0.92)	1.20	0.003	0.032
Lower abundance in the RP group
Q2KIS7	CLEC3B	Tetranectin	1.01 (0.76 − 1.37)	1.13 (0.803 − 1.25)	0.89	0.003	0.038
A0A4W2G034	SERPIND1	Serpin family D member 1	0.93 (0.90 − 1.08)	1.11 (1.03 − 1.18)	0.84	0.002	0.023
A0A4W2CCP9	APOA4	Apolipoprotein A4	0.80 (0.69 − 0.99)	1.16 (1.07 − 1.35)	0.69	0.004	0.010

FC– Median fold change between RP and PP groups, calculated as the ratio of median in RP group to median in PP group.

FDR– false discovery rate.

*−The BLAST analysis characterized the protein as Immunoglobulin heavy constant mu (Accession: A0A4W2DVR2; Gene: IGHM).

Figure 2. Gene Ontology for the proteins with the different abundance between the RP (n = 9) and PP (n = 6) groups using the PANTHER GO-Slim analysis: A) Molecular function; B) Cellular Component; C) Biological Process.

Table 2. Pathway enrichment analysis of the proteins differentially abundant between the group with retained placenta (RP; n = 9) and the group with the physiological puerperium (PP; n = 6). FDR < 0.05 is considered significant.

Pathway	FDR	Count		Genes
		Observed	Background
Platelet degranulation	<0.001	3	141	FGA, ITIH4, CLEC3B
Response to elevated platelet cytosolic Ca^2+	<0.001	3	148	FGA, ITIH4, CLEC3B
Hemostasis	<0.001	5	803	FGA, IGHM, ITIH4, CLEC3B, SERPIND1
Platelet activation, signaling and aggregation	0.002	3	293	FGA, ITIH4, CLEC3B
Common Pathway of Fibrin Clot Formation	0.003	2	25	FGA, SERPIND1
Assembly of active LPL and LIPC lipase complexes	0.004	1	30	APOA4
Formation of Fibrin Clot (Clotting Cascade)	0.007	2	43	FGA, SERPIND1
Plasma lipoprotein remodeling	0.010	1	56	APOA4
Detoxification of Reactive Oxygen Species	0.014	1	69	GPX3
Amyloid fiber formation	0.021	2	89	FGA, APOA4
Plasma lipoprotein assembly, remodeling, and clearance	0.022	1	98	APOA4
Scavenging of heme from plasma	0.022	2	106	HPX, HP
Post-translational protein phosphorylation	0.023	2	109	FGA, SERPIND1
Regulation of IGF transport and uptake by IGFBP	0.026	2	127	FGA, SERPIND1
Binding and Uptake of Ligands by Scavenger Receptors	0.045	2	168	HPX, HP

FDR–False discovery rate (FDR) P–value; Insulin-like Growth Factor–IGF; Insulin-like Growth Factor Binding Proteins–IGFBP; Observed count–The number of mapped identifiers that match the pathway for the selected molecular type; Background count–The total number of identifiers in the pathway for the selected molecular type.

Table 3. Validation results: protein, results (median and minimum–maximum range) in cows with retention of the placenta (RP; n = 9) and cows with the physiologic puerperium (PP; n = 6), and P–values indicating the significance of differences between the groups (Mann-Whitney U test, P–value < 0.05 is considered significant).

Protein (unit)	RP (n = 9)	PP (n = 6)	P‒value
HP (g/L)	0.46 (0.36‒1.33)	0.05 (0.05‒0.18)	0.001
LBP (µg/mL)	4.16 (0.86‒18.76)	0.63 (0.44‒0.75)	0.002
FGA (ng/mL)	8.6 (1.3‒266.8)	27.4 (8.8‒378.3)	0.195
GPX (U/L)	579 (448‒970)	516 (291‒798)	0.289
TNCT (ng/mL)	1.5 (0.5‒24.9)	3.7 (1.0‒47.2)	0.409

Abbreviations: FGA– fibrinogen alpha chain; GPX–glutathione peroxidase; HP–haptoglobin; LBP–lipopolysaccharide-binding protein; TNCT– tetranectin.

Table 4. Biochemistry parameters/ratios: reference intervals (Laboratory of the Internal Diseases Clinic, Faculty of Veterinary Medicine, University of Zagreb), results (median and minimum–maximum range) in cows with retention of the placenta (RP; n = 9) and cows with the physiologic puerperium (PP; n = 6), and P–values indicating the significance of differences between the groups (Man-Whitney U test, p < 0.05 is considered significant).

Parameter/ratio (unit)	Reference interval	RP (n = 9)	PP (n = 6)	P‒value
SAA (mg/L)	< 2	667 (390‒1687)	33 (0.1‒69)	0.002
GLU (mmol/L)	2.5‒4.2	3.3 (1.0‒3.4)	2.2 (1.1‒2.8)	0.036
Urea (mmol/L)	4.1‒5.8	2.5 (1.3‒4.0)	1.3 (1.1‒3.3)	0.087
CREAT (μmol/L)	< 133	94 (78‒151)	105 (65‒142)	0.814
T-BIL (μmol/L)	2.1‒7.7	5.7 (3.9‒11.5)	2.5 (2.0‒4.4)	0.007
TP (g/L)	60‒80	70 (68‒77)	70 (68‒76)	0.813
ALB (g/L)	30‒36	27 (25‒33)	28 (26‒36)	0.287
ALB/Urea (g/mmol)	N/A	11 (8‒19)	22 (11‒24)	0.018
Calcium (mmol/L)	2.50‒2.90	2.28 (2.00‒2.39)	2.15 (2.02‒2.28)	0.596
Magnesium (mmol/L)	0.70‒1.00	0.85 (0.74‒1.25)	1.02 (0.85‒1.96)	0.088
Phosphate (mmol/L)	1.60‒2.30	1.61 (1.39‒1.95)	2.42 (0.87‒2.83)	0.059
CHOL (mmol/L)	2.3‒4.7	2.1 (1.1‒2.4)	2.3 (2.0‒5.3)	0.409
TRIG (mmol/L)	0.06‒0.22	0.17 (0.15‒0.22)	0.17 (0.11‒0.17)	0.214
NEFA (mmol/L)	< 0.70	0.82 (0.54‒1.65)	0.45 (0.17‒1.24)	0.077
(NEFA + TRIG)/CHOL	N/A	0.85 (0.30‒0.90)	0.28 (0.06‒0.62)	0.025
BHB (mmol/L)	< 1.40	0.86 (0.56‒2.37)	0.99 (0.76‒2.89)	0.195
AST (U/L)	< 123	102 (55‒165)	90 (54‒99)	0.077
AST/BHB (U/mmol)	N/A	108 (70‒148)	71 (31‒100)	0.025
AST/CHOL (U/mmol)	N/A	68 (23‒85)	44 (10‒45)	0.025
GGT (U/L)	< 25	20 (14‒29)	29 (22‒31)	0.032
GLDH (U/L)	< 30	11 (4‒14)	6 (7‒17)	0.289
CK (U/L)	< 94	235 (53‒346)	87 (82‒160)	0.224
LDH (U/L)	692‒1445	938 (640‒1298)	838 (730‒890)	0.050

Abbreviations: ALB–albumin; AST–aspartate aminotransferase; BHB–β-hydroxybutyrate; CHOL– cholesterol; CREAT–creatinine; GGT–gamma-glutamyl transferase; GLDH–glutamate dehydrogenase; GLOB–globulins; GLU–glucose; LDH–lactate dehydrogenase; N/A–not available; NEFA–non-esterified fatty acids; SAA–serum amyloid A; T-BIL–total bilirubin; TRIG–triglycerides; TP–total protein.

Table 5. Results of the ROC analysis for the routine biochemistry parameters with levels differing between cows with retention of the placenta (n = 9) and cows with the physiologic puerperium (n = 6): Area Under the Curve (AUC) with 95% Confidence Interval (CI), P-value, Cut-off, sensitivity (95% CI), and specificity (95% CI).

Parameter/ratio (unit)	AUC	P‒value	Cut-off	Sensitivity (%)	Specificity (%)
SAA (mg/L)	1.000 (0.782‒1.000)	<0.001	69	100 (66‒100)	100 (54‒100)
GLU (mmol/L)	0.861 (0.589‒0.982)	0.004	2.8	67 (30‒92)	100 (54‒100)
T-BIL (μmol/L)	0.926 (0.670‒0.997)	<0.001	4.4	78 (40‒97)	100 (54‒100)
ALB/Urea (g/mmol)	0.833 (0.556‒0.971)	0.001	19.2	100 (66‒100)	67 (22‒96)
(NEFA + TRIG)/CHOL	0.852 (0.578‒0.978)	0.002	0.29	100 (66‒100)	67 (22‒96)
AST/BHB (U/mmol)	0.852 (0.578‒0.978)	0.002	100	67 (30‒92)	100 (54‒100)
AST/CHOL (U/mmol)	0.852 (0.578‒0.978)	0.001	44	78 (40‒97)	100 (54‒100)
GGT (U/L)	0.833 (0.556‒0.971)	0.001	21	67 (30‒92)	100 (54‒100)

Abbreviations: ALB–albumin; AST–aspartate aminotransferase; BHB–β-hydroxybutyrate; CHOL– cholesterol; CREAT–creatinine; GGT–gamma-glutamyl transferase; GLDH–glutamate dehydrogenase; GLOB–globulins; GLU–glucose; LDH–lactate dehydrogenase; N/A–not available; NEFA–non-esterified fatty acids; SAA–serum amyloid A; T-BIL–total bilirubin; TRIG–triglycerides; TP–total protein.

Figure 3. A graphical representation of the relationship between the number of altered proteins and serum amyloid A (SAA) concentration in serum.

Table 6. Correlation between the number of proteins with the altered abundance and the routine biochemistry parameters: Spearman’s rho rank correlation coefficient (ρ) with the 95% confidence interval and P–value.

Parameter/ratio (unit)	ρ	P‒value
SAA (mg/L)	0.913 (0.752 to 0.971)	<0.001
GLU (mmol/L)	0.359 (-0.187 to 0.736)	0.188
Urea (mmol/L)	0.460 (-0.069 to 0.787)	0.084
CREAT (μmol/L)	−0.189 (-0.639 to 0.358)	0.500
T-BIL (μmol/L)	0.867 (0.637 to 0.955)	<0.001
TP (g/L)	0.193 (-0.354 to 0.642)	0.490
ALB (g/L)	−0.066 (-0.560 to 0.462)	0.815
ALB/Urea (g/mmol)	−0.591 (-0.847 to −0.113)	0.020
Calcium (mmol/L)	0.038 (-0.484 to 0.540)	0.892
Magnesium (mmol/L)	−0.232 (-0.665 to 0.318)	0.405
Phosphate (mmol/L)	−0.412 (-0.763 to 0.127)	0.127
CHOL (mmol/L)	−0.067 (-0.560 to 0.461)	0.812
TRIG (mmol/L)	0.595 (0.119 to 0.849)	0.019
NEFA (mmol/L)	0.586 (0.105 to 0.844)	0.022
(NEFA + TRIG)/CHOL	0.617 (0.153 to 0.858)	0.014
BHB (mmol/L)	−0.129 (-0.602 to 0.410)	0.646
AST (U/L)	0.516 (0.006 to 0.813)	0.049
AST/BHB (U/mmol)	0.400 (-0.142 to 0.757)	0.140
AST/CHOL (U/mmol)	0.428 (-0.107 to 0.771)	0.111
GGT (U/L)	−0.277 (-0.691 to 0.274)	0.317
GLDH (U/L)	−0.091 (-0.577 to 0.442)	0.747
CK (U/L)	0.349 (-0.199 to 0.730)	0.203
LDH (U/L)	0.428 (-0.107 to 0.771)	0.111