Comparative Analysis of Serum Proteins Between Hepatitis B Virus Genotypes B and C Infection by DIA-Based Quantitative Proteomics

Figures & data

Table 1 Baseline Characteristics of Patients Enrolled in This Study

Characteristic	Health Controls (n = 18)	HBV-B Group (n = 18)	HBV-C Group (n = 18)
Gender (female/male)	9/9	12/6	12/6
Age (yrs.)	39.17 ± 8.28	44.50 ±10.12	39.39 ± 10.80
ALT (U/L)	30.78 ± 4.68	573.22 ± 446.4*	204.44 ± 216.29§
AST (U/L)	24.72 ± 6.43	337.39 ± 319.01*	149.72 ± 159.75§
ALP (U/L)	99.17 ± 28.81	101.89 ± 38.84	87.56 ± 26.94
GGT (U/L)	40.94 ± 10.34	116.83 ± 103.35*	69.22 ± 46.07§
PLT (10^9 L^−1)	132.56 ± 11.52	150.50 ± 42.03	152.39 ± 59.24
TB (umol/L)	7.90 ± 0.83	45.49 ± 79.48*	27.62 ± 34.72§
TBA (umol/L)	9.76 ± 14.13	49.81 ± 69.47*	44.41 ± 70.04
ALB (g/L)	41.36 ± 3.21	41.56 ± 6.10	39.57 ± 10.52
APRI	0.47 ± 0.11	6.40 ± 6.31*	3.53 ± 5.04§
PT (s)	12.69 ± 0.94	16.00 ± 5.77*	14.31 ± 1.58
APTT (s)	35.46 ± 4.46	40.31 ± 4.49	38.82 ± 4.04
FIB (g/L)	2.58 ± 0.38	2.49 ± 0.64	2.72 ± 0.62
HBeAg	NA	139.90 ± 343.30	436.95 ± 527.10§
HBV viral load (log10)	NA	6.31± 1.62	6.87± 1.56

Notes: Data are means ± SD; P < 0.05 for comparisons between HBV-B and healthy controls*, and between HBV-C and HBV-B§.

Abbreviations: ALT, alanine transferase; AST, aspartate transferase; ALP, alkaline phosphatase; GGT, γ-Glutamyl transferase; PLT, platelet; TB, total bilirubin; TBA, total bile acids; ALB, albumin; APRI, aspartate aminotransferase -to- platelet ratio index; PT, prothrombin time; APTT, activated partial thromboplasting time; FIB, fibrinogen; HBeAg, hepatitis B e antigen.

Figure 1 Identification of serum proteins in HBV patients infected with genotype B and those infected genotype C. (A) The number of identified peptides in 3 repeated experiments. (B) The number of identified protein groups in 3 repeated experiments. (C)The Venn diagrams show the numbers of identified proteins and the overlaps of protein identification in the 3 groups.

Table 2 List of the Typically Differentially Expressed Proteins Between the Serum Samples from HBV Patients Infected with Genotype B and Those Infected Genotype C

Protein Accession	Protein Description	Gene Name	Fold Change HBV-B/Healthy Controls	Fold Change HBV-B/Healthy Controls	Fold Change HBV-B/Healthy Controls
A0A1B1CYC5	Vitamin D binding protein (Fragment)	Gc	0.24	1.81	7.50
A0A1B0GTC3	Alpha-ketoglutarate-dependent dioxygenase FTO	FTO	0.21	0.91	4.28
P05546	Heparin cofactor 2	SERPIND1	0.34	0.80	2.35
P48740	Mannan-binding lectin serine protease 1	MASP1	0.34	0.76	2.25
B7Z550	Complement component 8, beta polypeptide, isoform CRA_b	C8B	0.55	1.12	2.02
P35527	Keratin, type I cytoskeletal 9	KRT9	11.41	19.77	1.73
P04275	von Willebrand factor	VWF	2.52	4.21	1.67
A3KPE2	Apolipoprotein C-III, isoform CRA_a	APOC3	0.55	0.71	1.29
P15814	Immunoglobulin lambda-like polypeptide 1	IGLL1	2.45	1.90	0.77
K7ER74	APOC4-APOC2 readthrough (NMD candidate)	APOC4-APOC2	0.59	0.45	0.76
A0A0C4DH63	T cell receptor gamma joining P (Fragment)	TRGJP	2.95	2.04	0.69
P19320	Vascular cell adhesion protein 1	VCAM1	17.63	7.41	0.42
A0A1W2PQB1	Fc of IgG low affinity IIIa receptor isoform 1	FCGR3A	7.74	2.27	0.29
A2VCK8	Thymosin beta 4, X-linked	TMSB4X	1.44	0.41	0.28
A0A0A0MR13	Guanine nucleotide-binding protein G(s) subunit alpha isoforms short	GNAS	0.21	0.05	0.25
Q65ZC9	Single-chain Fv (Fragment)	scFv	13.90	2.71	0.19
Q5CZ94	Uncharacterized protein DKFZp781M0386	DKFZp781M0386	3.85	0.58	0.15
A0A0G2JS06	Immunoglobulin lambda variable 5–39	IGLV5-39	4.65	0.47	0.10

Figure 2 Bioinformatics analysis of differentially expressed proteins between the serum samples of the healthy controls and those of HBV-genotype B infected patients. (A) Volcano plot representing the protein abundance changes (groups B vs A). A total of 53 dysregulated proteins with fold change ≥±1.5 and p-values < 0.05 were identified. (B) Hierarchical clustering of the 53 dysregulated proteins (groups B vs A). (C) GO analysis of 53 dysregulated proteins (groups B vs A). The abscissa represents enriched GO function classifications, which were divided into three major categories: biological process (BP), molecular function (MF) and cellular component (CC). (D) KOG analysis of 53 dysregulated proteins (groups B vs A).

Figure 3 Bioinformatics analysis of differentially expressed proteins between the serum samples of the healthy controls and those of HBV-genotype C infected patients. (A) Volcano plot representing the protein abundance changes (groups C vs A). A total of 59 dysregulated proteins with fold change ≥±1.5 and p-values < 0.05 were identified. (B) Hierarchical clustering of the 66 dysregulated proteins (groups C vs A). (C) GO analysis of 59 dysregulated proteins (groups C vs A). The abscissa represents enriched GO function classifications, which were divided into three major categories: biological process (BP), molecular function (MF) and cellular component (CC). (D) KOG analysis of 59 dysregulated proteins (groups C vs A).

Figure 4 KEGG annotation of the dysregulated proteins from groups of healthy control individuals and HBV-B (A) and groups of healthy control individuals and HBV-C (B).

Figure 5 Key signaling pathways involved in the serum samples from HBV patients infected with genotype B and those infected genotype C. ECM–receptor interaction (A) and complement and coagulation cascades (B) obtained from KEGG pathway-based enrichment analysis of dysregulated proteins.

Figure 6 Validation of the selected differentially expressed proteins of VWF (A) and C8B (B) in the serum samples from HBV patients infected with genotype B and those infected genotype C by ELISA in the validation cohort. Data are expressed as the mean SEM (n = 36, *P < 0.05, **P < 0.01).