# iTRAQ-Based Proteomics Identification of Serum Biomarkers of Two Chronic Hepatitis B Subtypes Diagnosed by Traditional Chinese Medicine

Authors: Jiankun Yang; Lichao Yang; Baixue Li; Weilong Zhou; Sen Zhong; Zhenhua Zhuang; Bin Yang; Maoshan Chen; Quansheng Feng
Journal: BioMed Research International
 (2016)

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## Abstract

Background. Chronic infection with hepatitis B virus (HBV) is a leading cause of cirrhosis and hepatocellular carcinoma. By traditional Chinese medicine (TCM) pattern classification, damp heat stasis in the middle-jiao (DHSM) and liver Qi stagnation and spleen deficiency (LSSD) are two most common subtypes of CHB.Results. In this study, we employed iTRAQ proteomics technology to identify potential serum protein biomarkers in 30 LSSD-CHB and 30 DHSM-CHB patients. Of the total 842 detected proteins, 273 and 345 were differentially expressed in LSSD-CHB and DHSM-CHB patients compared to healthy controls, respectively. LSSD-CHB and DHSM-CHB shared 142 upregulated and 84 downregulated proteins, of which several proteins have been reported to be candidate biomarkers, including immunoglobulin (Ig) related proteins, complement components, apolipoproteins, heat shock proteins, insulin-like growth factor binding protein, and alpha-2-macroglobulin. In addition, we identified that proteins might be potential biomarkers to distinguish LSSD-CHB from DHSM-CHB, such as A0A0A0MS51_HUMAN (gelsolin), PON3_HUMAN, Q96K68_HUMAN, and TRPM8_HUMAN that were differentially expressed exclusively in LSSD-CHB patients and A0A087WT59_HUMAN (transthyretin), ITIH1_HUMAN, TSP1_HUMAN, CO5_HUMAN, and ALBU_HUMAN that were differentially expressed specifically in DHSM-CHB patients.Conclusion. This is the first time to report serum proteins in CHB subtype patients. Our findings provide potential biomarkers can be used for LSSD-CHB and DHSM-CHB.


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## Body

## 1. Introduction

Chronic hepatitis B virus (CHB) infection is a leading cause of cirrhosis and hepatocellular carcinoma (HCC) and, in addition to morbidity and mortality, creates significant economic and social burdens [1, 2]. It is estimated that approximately 240 million people have CHB infection worldwide and CHB infection should be responsible for 650,000 cases of hepatocellular carcinoma [2, 3]. Due to the pathogenicity of CHB, early detection of CHB infection is the goal of treatment to diagnose and prevent the progression [4]. To this end, several hepatitis B virus (HBV) markers have been identified, including antigens (hepatitis B surface antigen, HBsAg; hepatitis Be antigen, HBeAg; hepatitis B core antigen, HBcAg), antibodies (hepatitis surface antibody, anti-HBs; hepatitis Be antibody, anti-HBe; hepatitis B core antibody, anti-HBc), and immunoglobulin (Ig) G and immunoglobulin M; however, unequivocal diagnosis requires more biomarkers [5].By traditional Chinese medicine (TCM) pattern classification, CHB infected patients are accordingly classified into six subtypes [6]: (1) damp heat stasis in the middle-jiao (DHSM), (2) liver Qi stagnation and spleen deficiency (LSSD), (3) Yang deficiency of spleen and kidney (YDSK), (4) Yin deficiency of liver and kidney (YDLK), (5) blood stasis into collateral (BSIC), and (6) damp heat complicated with blood stasis (DHBS). Among them DHSM and LSSD are two most common CHB subtypes and have unique syndromes in clinic. For example, LSSD patients always have main syndromes, such as (Mi) flank pain and (Mii) abdominal distension and loose stools, and secondary symptoms, including (Si) depression and boredom, (Sii) body tired fatigue, and (Siii) pale tongue with teeth marks. DHSM patients have another two main syndromes, such as (M1) abdominal distension and (M2) yellow greasy moss, and three secondary syndromes, including (S1) nausea, being tired of the oil, and poor appetite, (S2) jaundice, bright color, and dark urine, and (S3) viscous stool foul smell. However, these syndromes are diagnosed by TCM doctors according to their experiences and the molecular biomarkers remain unclear.Proteomics is a powerful technology recently developed to enhance our study on the diagnosis, treatment, and prevention of human diseases [7]. Among the proteomics technologies iTRAQ (isobaric Tags for Relative and Absolute Quantitation) has become popular for protein identification and quantification due to its sensitivity, accuracy, and high throughput [8]. It has been used to identify biomarker proteins for different stages of hepatitis B related diseases in patients and cellular models [9–12]. Several serum proteins have been reported to be potential biomarkers for CHB, such as actin [13], apolipoproteins A-I and A-IV [14], complement component [15], immunoglobulin related proteins [15, 16], haptoglobins β and α2 chain [14], and transferrin [17].In this study, we employed iTRAQ combined with LC-ESI-MS/MS analyses to investigate protein biomarkers in the serum samples of two CHB subtype patients (LSSD and DHSM). Compared to healthy controls we found a number of proteins differentially expressed in both LSSD and DHSM CHB subtypes, such as actin, apolipoprotein, complement component, and immunoglobulin related proteins. In addition, we identified some proteins differentially expressed exclusively in one of LSSD and DHSM groups, such as gelsolin (GSN), likely SNC73 protein, and transient receptor potential cation channel subfamily M member 8 (TRPM8) that were found with different expression in LSSD-CHB patients only and transthyretin (TTR), tubulin, and keratin types I and II that were differentially expressed in DHSM-CHB patients only. Our findings not only validate previously reported CHB protein biomarkers but also report for the first time protein biomarkers for LSSD and DHSM CHB subtypes. The output of this study gives a valuable resource for future HBV associated studies and provides new insights of traditional Chinese medicine on molecular level.


## 2. Materials and Methods

### 2.1. Ethics Statement

This study was conducted in compliance with the Declaration of Helsinki, the ethics approval was granted by the research medical ethics committee of Chengdu University of Traditional Chinese Medicine, and signed informed consent was obtained from all participants.


### 2.2. Patients and Serum Collection

A total of 104 CHB patients were enrolled from West China Hospital, Sichuan University, and filtered with strict clinical evaluation described below. For iTRAQ proteomics analysis, we obtained blood samples from 30 LSSD-CHB patients, 30 DHSM-CHB patients, and 20 healthy controls (HCTL). For western blot analysis, 9 LSSD-CHB patients, 9 DHSM-CHB patients, and 6 HCTL participants were enrolled. Serum was collected from blood sample (4 mL) following the manufacture’s protocol. Briefly, blood sample was incubated at room temperature for 2 h in vacutainer blood handling tube (Becton Dickinson, New Jersey, USA) and centrifuged for 10 min at 3,000 rpm and 4°C. Serum sample, which is the result supernatant, was transferred into a clean polypropylene tube and stored at −80°C.


### 2.3. Clinical Evaluation

The viral markers HBsAg, HBeAg, anti-HBs, anti-HBc, and anti-HBe were determined routinely in serum samples using standard procedures (AxSYM®; Abbott Laboratories, Rungis, France), as well as other molecular diagnostic markers like ALT (alanine transaminase), AST (aspartate aminotransferase), STB (serum total bilirubin), CB (conjugated bilirubin), UCB (unconjugated bilirubin), and HBV-DNA. Participants, who have hepatitis B history or HBsAg positive history for more than six months, were diagnosed as chronic HBV infection if they were positive to HBsAg and/or HBV-DNA. We used both western and Chinese medicine criteria to divide CHB patients into two groups. First, participants were satisfied with the following requirements: (1) serum HBsAg positive for over 6 months; (2) HBV-DNA positive; (3) continuous or repeated elevated serum ALT in last 12 months; (4) being 18~60 years old; (5) no planed move during the test. Then, LSSD-CHB and DHSM-CHB patients were diagnosed using the clinical symptoms mentioned before. CHB patients were diagnosed as LSSD-CHB when they met the criteria: (1) Mi and Mii; (2) Mi, Sii, and Siii; (3) Mii and Si. DHSM-CHB patients were diagnosed as follows: (1) M1 and M2; (2) M1, S1, and S2; (3) M2 and two of the secondary symptoms. We also filtered the patients when they satisfied one of the following criteria: (1) being associated with other types of hepatitis viruses or human immunodeficiency virus (HIV); (2) cirrhosis, malignancy; (3) being diagnosed with fulminant hepatitis (including acute, subacute, and chronic severe hepatitis); (4) being associated with drug or toxic liver, autoimmune hepatitis, and genetic-metabolic liver disease; (5) heart, lung, kidney, endocrine, blood, and other serious diseases; (6) pregnant women and lactating women; (7) mental disorders, in line with Chinese Classification of Mental Disorders Diagnosis (CCMD-3) standard; (8) other individuals not suitable for the cohort study.


### 2.4. Protein Preparation

Serum sample (200μL) from each patient was processed to reduce the complexity by using ProteoMiner™ Kits (Bio-Rad Laboratories, Hercules, CA, USA). Then, the sample was eluted using Lysis buffer at pH 8.5 (2M Thiourea, 7M Urea, 4% CHAPS, and 40 mM Tris-HCl), reduced using 10 mM DTT at 56°C for 1 h, and alkylated using 55 mM IAM in darkness for 1 h. After being precipitated within chilled acetone (4 × volume) at −20°C overnight, the protein sample was centrifuged at 30,000 ×g for 15 min at 4°C; the pellet was next dissolved in 500 μL of 0.5 M triethylammonium bicarbonate (Applied Biosystems, Milan, Italy) and sonicated at 200 W in ice for 15 min. Finally, the samples were centrifuged again at 30,000 ×g for 15 min at 4°C, and the supernatant was quantified using Bradford Protein Assay Kit (CWBIO, Beijing, China) and stored at −80°C for subsequent analysis.


### 2.5. iTRAQ Sample Labelling, SCX Fractionation, and LC-ESI-MS/MS Analysis

Proteins isolated from 10 individuals in the same group were pooled for iTRAQ labelling. Pooled protein samples (100μg) were digested using Trypsin Gold (Promega, Madison, WI, USA) at 37°C for 16 h (protein: trypsin = 30 : 1). Digested peptides were dried by vacuum centrifugation, reconstituted in 0.5 M triethylammonium bicarbonate (Applied Biosystems, Milan, Italy), and processed 8-plex iTRAQ (Applied Biosystems) labelling following the protocols. Samples were labelled with the iTRAQ tags as follows: LSSD-CHB (113, 115, and 117), DHSM-CHB (114, 116, and 118), and HCTL (119 and 121). After being incubated at room temperature for 2 h, all the peptide mixtures were then pooled and dried by vacuum centrifugation. Strong cation exchange (SCX) chromatography was performed using the LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan), as previously described [18].SFX fractions were resuspended in buffer A (2% ACN, 0.1% FA), followed by a centrifugation at 20,000 g for 10 min. Then, 10μL of the supernatant was loaded onto a 2 cm C18 trap column on a LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan) by the autosampler and eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house. At 300 nL/min the samples were loaded with buffer B (98% ACN, 0.1% FA) as the following procedural: 5% B for 1 min, a 44 min gradient from 2 to 35% B, a 2 min linear gradient to 80%, and 80% B for 4 min. Finally, the chromatographic conditions were restored in 1 min. Data acquisition was performed with an Q EXACTIVE (Thermo Fisher Scientific, San Jose, CA) coupled online to the HPLC, as described [19, 20].


### 2.6. Database Search and Protein Quantification

Database search and protein quantification were performed using Mascot (Matrix Science, London, UK; version 2.4.0). Briefly, raw data files acquired from the Orbitrap were converted into mascot generic format (MGF) files using msconvert tool of ProteoWizard (http://proteowizard.sourceforge.net/) [21]. To identify expressed proteins and quantify them, all 20 MGF files were merged and searched against UniProtHUMAN (2016_04, 152,544 sequences) database using Mascot with parameters: quantification: iTRAQ 8plex (Applied Biosystems iTRQA™ 8-plex); enzyme: trypsin; fixed modification: carboxymethyl (C), iTRAQ8plex (N-term) and iTRAQ8plex (K); variable modifications: dioxidation (M), oxidation (M), and iTRAQ8plex (Y), mass values: monoisotopic; peptide mass tolerance: ±15 ppm; fragment mass tolerance: ±20 mmu; max missed cleavages: (1) The charge states of peptides were set to +2 and +3. Specifically, an automatic decoy database search was performed in Mascot by choosing the decoy checkbox in which a random sequence of database is generated and tested for raw spectra as well as the real database. To reduce the probability of false peptide identification, only peptides at the 95% confidence interval by a Mascot probability analysis greater than “identity” were counted as identified. And each confident protein identification involves at least one unique peptide.


### 2.7. Protein Different Expression and Functional Analysis

To identify differentially expressed proteins in LSSD-CHB and DHSM-CHB compared to HCTL, we set a cut-off for fold change (>1.2) of protein abundance provided by Mascot andp value (<0.05) calculated by edgeR [22]. Venn diagram of up- and downregulated proteins was analyzed by InteractiVenn (http://www.interactivenn.net/) [23]. To annotate potential functions of proteins, UniProt IDs of candidate proteins were submitted to DAVID Bioinformatics Resources 6.7 (https://david.ncifcrf.gov/home.jsp) [24] and STRING v10 (http://string-db.org/) [25], Gene Ontology (GO), and KEGG pathway were selected, and we used false discovery rate (FDR) to control the results. Protein-protein interaction networks were analyzed by STRING.


### 2.8. Western Blot Analysis

Protein samples obtained from serum of 9 LSSD-CHB patients, 9 DHSM-CHB patients, and 6 healthy individuals were resolved by 12% SDS-PAGE using Miniprotean II electrophoresis unit (Bio-Rad) run at constant 120 V for 1 h and transferred to a PVDF membrane (Amersham Biosciences) under a constant voltage of 15 V for 20 min. The membranes were blocked with 5% skim milk powder in Tris-buffered saline with 0.05% Tween-20 (TTBS) for 1 h and probed in TTBS with primary antibodies (1 : 500, Santa Cruz Biotechnology, CA, USA), anti-PSMA7 (sc-166761), anti-PF4V (sc-367359), anti-PSMA6 (sc-271187), anti-SERPING1 (sc-377062), anti-ACTB (sc-8432), anti-AHSG (sc-137102), anti-CTSC (sc-74590), anti-PLTP (sc-271596), and anti-ALB (sc-46293), followed by incubation with secondary antibody (1 : 1000) for 1 h in darkness. All antibody incubations were carried out using gentle orbital shaking at room temperature. Western blots were washed five times in TTBS (5 min × 2 and 10 min × 3) after each incubation step and visualized with enhanced chemiluminescence (ECL, GE Healthcare) following the manufacturers’ instructions. Band intensities on the Western blots were quantified using ImageJ (Wayne Rasband, National Institutes of Health). Albumin was used as reference to calculate the relative intensity of each protein. Then, mean ± SD values of each protein in HCTL and patients were calculated and compared using GraphPad Prism (http://www.graphpad.com/).


### 2.9. Statistical Analysis

Statistical analysis including the calculation of mean value, standard deviation (SD), and students’t-test was performed by using GraphPad Prism (v 6.02). The Holm-Sidak method was used to calculate the statistical significance of multiple clinical diagnostic values.


## 2.1. Ethics Statement

This study was conducted in compliance with the Declaration of Helsinki, the ethics approval was granted by the research medical ethics committee of Chengdu University of Traditional Chinese Medicine, and signed informed consent was obtained from all participants.


## 2.2. Patients and Serum Collection

A total of 104 CHB patients were enrolled from West China Hospital, Sichuan University, and filtered with strict clinical evaluation described below. For iTRAQ proteomics analysis, we obtained blood samples from 30 LSSD-CHB patients, 30 DHSM-CHB patients, and 20 healthy controls (HCTL). For western blot analysis, 9 LSSD-CHB patients, 9 DHSM-CHB patients, and 6 HCTL participants were enrolled. Serum was collected from blood sample (4 mL) following the manufacture’s protocol. Briefly, blood sample was incubated at room temperature for 2 h in vacutainer blood handling tube (Becton Dickinson, New Jersey, USA) and centrifuged for 10 min at 3,000 rpm and 4°C. Serum sample, which is the result supernatant, was transferred into a clean polypropylene tube and stored at −80°C.


## 2.3. Clinical Evaluation

The viral markers HBsAg, HBeAg, anti-HBs, anti-HBc, and anti-HBe were determined routinely in serum samples using standard procedures (AxSYM®; Abbott Laboratories, Rungis, France), as well as other molecular diagnostic markers like ALT (alanine transaminase), AST (aspartate aminotransferase), STB (serum total bilirubin), CB (conjugated bilirubin), UCB (unconjugated bilirubin), and HBV-DNA. Participants, who have hepatitis B history or HBsAg positive history for more than six months, were diagnosed as chronic HBV infection if they were positive to HBsAg and/or HBV-DNA. We used both western and Chinese medicine criteria to divide CHB patients into two groups. First, participants were satisfied with the following requirements: (1) serum HBsAg positive for over 6 months; (2) HBV-DNA positive; (3) continuous or repeated elevated serum ALT in last 12 months; (4) being 18~60 years old; (5) no planed move during the test. Then, LSSD-CHB and DHSM-CHB patients were diagnosed using the clinical symptoms mentioned before. CHB patients were diagnosed as LSSD-CHB when they met the criteria: (1) Mi and Mii; (2) Mi, Sii, and Siii; (3) Mii and Si. DHSM-CHB patients were diagnosed as follows: (1) M1 and M2; (2) M1, S1, and S2; (3) M2 and two of the secondary symptoms. We also filtered the patients when they satisfied one of the following criteria: (1) being associated with other types of hepatitis viruses or human immunodeficiency virus (HIV); (2) cirrhosis, malignancy; (3) being diagnosed with fulminant hepatitis (including acute, subacute, and chronic severe hepatitis); (4) being associated with drug or toxic liver, autoimmune hepatitis, and genetic-metabolic liver disease; (5) heart, lung, kidney, endocrine, blood, and other serious diseases; (6) pregnant women and lactating women; (7) mental disorders, in line with Chinese Classification of Mental Disorders Diagnosis (CCMD-3) standard; (8) other individuals not suitable for the cohort study.


## 2.4. Protein Preparation

Serum sample (200μL) from each patient was processed to reduce the complexity by using ProteoMiner™ Kits (Bio-Rad Laboratories, Hercules, CA, USA). Then, the sample was eluted using Lysis buffer at pH 8.5 (2M Thiourea, 7M Urea, 4% CHAPS, and 40 mM Tris-HCl), reduced using 10 mM DTT at 56°C for 1 h, and alkylated using 55 mM IAM in darkness for 1 h. After being precipitated within chilled acetone (4 × volume) at −20°C overnight, the protein sample was centrifuged at 30,000 ×g for 15 min at 4°C; the pellet was next dissolved in 500 μL of 0.5 M triethylammonium bicarbonate (Applied Biosystems, Milan, Italy) and sonicated at 200 W in ice for 15 min. Finally, the samples were centrifuged again at 30,000 ×g for 15 min at 4°C, and the supernatant was quantified using Bradford Protein Assay Kit (CWBIO, Beijing, China) and stored at −80°C for subsequent analysis.


## 2.5. iTRAQ Sample Labelling, SCX Fractionation, and LC-ESI-MS/MS Analysis

Proteins isolated from 10 individuals in the same group were pooled for iTRAQ labelling. Pooled protein samples (100μg) were digested using Trypsin Gold (Promega, Madison, WI, USA) at 37°C for 16 h (protein: trypsin = 30 : 1). Digested peptides were dried by vacuum centrifugation, reconstituted in 0.5 M triethylammonium bicarbonate (Applied Biosystems, Milan, Italy), and processed 8-plex iTRAQ (Applied Biosystems) labelling following the protocols. Samples were labelled with the iTRAQ tags as follows: LSSD-CHB (113, 115, and 117), DHSM-CHB (114, 116, and 118), and HCTL (119 and 121). After being incubated at room temperature for 2 h, all the peptide mixtures were then pooled and dried by vacuum centrifugation. Strong cation exchange (SCX) chromatography was performed using the LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan), as previously described [18].SFX fractions were resuspended in buffer A (2% ACN, 0.1% FA), followed by a centrifugation at 20,000 g for 10 min. Then, 10μL of the supernatant was loaded onto a 2 cm C18 trap column on a LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan) by the autosampler and eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house. At 300 nL/min the samples were loaded with buffer B (98% ACN, 0.1% FA) as the following procedural: 5% B for 1 min, a 44 min gradient from 2 to 35% B, a 2 min linear gradient to 80%, and 80% B for 4 min. Finally, the chromatographic conditions were restored in 1 min. Data acquisition was performed with an Q EXACTIVE (Thermo Fisher Scientific, San Jose, CA) coupled online to the HPLC, as described [19, 20].


## 2.6. Database Search and Protein Quantification

Database search and protein quantification were performed using Mascot (Matrix Science, London, UK; version 2.4.0). Briefly, raw data files acquired from the Orbitrap were converted into mascot generic format (MGF) files using msconvert tool of ProteoWizard (http://proteowizard.sourceforge.net/) [21]. To identify expressed proteins and quantify them, all 20 MGF files were merged and searched against UniProtHUMAN (2016_04, 152,544 sequences) database using Mascot with parameters: quantification: iTRAQ 8plex (Applied Biosystems iTRQA™ 8-plex); enzyme: trypsin; fixed modification: carboxymethyl (C), iTRAQ8plex (N-term) and iTRAQ8plex (K); variable modifications: dioxidation (M), oxidation (M), and iTRAQ8plex (Y), mass values: monoisotopic; peptide mass tolerance: ±15 ppm; fragment mass tolerance: ±20 mmu; max missed cleavages: (1) The charge states of peptides were set to +2 and +3. Specifically, an automatic decoy database search was performed in Mascot by choosing the decoy checkbox in which a random sequence of database is generated and tested for raw spectra as well as the real database. To reduce the probability of false peptide identification, only peptides at the 95% confidence interval by a Mascot probability analysis greater than “identity” were counted as identified. And each confident protein identification involves at least one unique peptide.


## 2.7. Protein Different Expression and Functional Analysis

To identify differentially expressed proteins in LSSD-CHB and DHSM-CHB compared to HCTL, we set a cut-off for fold change (>1.2) of protein abundance provided by Mascot andp value (<0.05) calculated by edgeR [22]. Venn diagram of up- and downregulated proteins was analyzed by InteractiVenn (http://www.interactivenn.net/) [23]. To annotate potential functions of proteins, UniProt IDs of candidate proteins were submitted to DAVID Bioinformatics Resources 6.7 (https://david.ncifcrf.gov/home.jsp) [24] and STRING v10 (http://string-db.org/) [25], Gene Ontology (GO), and KEGG pathway were selected, and we used false discovery rate (FDR) to control the results. Protein-protein interaction networks were analyzed by STRING.


## 2.8. Western Blot Analysis

Protein samples obtained from serum of 9 LSSD-CHB patients, 9 DHSM-CHB patients, and 6 healthy individuals were resolved by 12% SDS-PAGE using Miniprotean II electrophoresis unit (Bio-Rad) run at constant 120 V for 1 h and transferred to a PVDF membrane (Amersham Biosciences) under a constant voltage of 15 V for 20 min. The membranes were blocked with 5% skim milk powder in Tris-buffered saline with 0.05% Tween-20 (TTBS) for 1 h and probed in TTBS with primary antibodies (1 : 500, Santa Cruz Biotechnology, CA, USA), anti-PSMA7 (sc-166761), anti-PF4V (sc-367359), anti-PSMA6 (sc-271187), anti-SERPING1 (sc-377062), anti-ACTB (sc-8432), anti-AHSG (sc-137102), anti-CTSC (sc-74590), anti-PLTP (sc-271596), and anti-ALB (sc-46293), followed by incubation with secondary antibody (1 : 1000) for 1 h in darkness. All antibody incubations were carried out using gentle orbital shaking at room temperature. Western blots were washed five times in TTBS (5 min × 2 and 10 min × 3) after each incubation step and visualized with enhanced chemiluminescence (ECL, GE Healthcare) following the manufacturers’ instructions. Band intensities on the Western blots were quantified using ImageJ (Wayne Rasband, National Institutes of Health). Albumin was used as reference to calculate the relative intensity of each protein. Then, mean ± SD values of each protein in HCTL and patients were calculated and compared using GraphPad Prism (http://www.graphpad.com/).


## 2.9. Statistical Analysis

Statistical analysis including the calculation of mean value, standard deviation (SD), and students’t-test was performed by using GraphPad Prism (v 6.02). The Holm-Sidak method was used to calculate the statistical significance of multiple clinical diagnostic values.


## 3. Results

### 3.1. Diagnosis of the Patients

To study serum protein biomarkers in LSSD and DHSM CHB patients, we obtained a total of 80 participants, including 30 LSSD-CHB, 30 DHSM-CHB patients, and 20 healthy volunteers. As shown in Table1 and Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/3290260, mean ages of LSSD-CHB, DHSM-CHB, and HCTL were 30, 36.8, and 35.5 years, respectively. Except missing information of three, all patients were positive to HBsAg and anti-HBc. There were two LSSD-CHB patients positive to anti-HBs, and 9 LSSD-CHB and 12 DHSM-CHB patients positive to anti-HBe. HBV-DNA levels in the blood samples of LSSD-CHB and DHSM-CHB patients were ranged from 5.12E+03~1.12E+08 IU/mL and 6.34E+04~9.40E+08 IU/mL, respectively. It is interesting that hepatitis B viral load (HBV-DNA copies) was significantly different (p=0.0096) in LSSD-CHB and DHSM-CHB patients. Next, we examined ALT, AST, STB, CB, and UCB levels in the blood samples of CHB patients. Mean values of these diagnosis biomarkers in the blood samples of LSSD-CHB and DHSM-CHB patients were similar. In addition, the levels of ALT and AST remained at a high level, compared to healthy individuals [26, 27], which confirmed their CHB infection.Table 1
Clinical diagnosis of patients who participated in this study.
Diagnosis Unit LSSD-CHB (n=30) DHSM-CHB (n=30) HCTL (n=20) p value Sex Male  24 15 10 Female  6 15 10  Age Years 17~56 18~60 24~56 Mean age 30.8 36.83 36.15 Standard deviation (SD) 10.526 11.885 11.554  Hepatitis B surface antigen (HBsAg) Positive  28 29 Negative  0 0  Hepatitis surface antibody (anti-HBs) Positive  2 0 Negative  25 29  Hepatitis Be antigen (HBeAg) Positive  21 19 Negative  7 9  Hepatitis Be antibody (anti-HBe) Positive  9 12 Negative  19 16  Hepatitis B core antibody (anti-HBc) Positive  28 29 Negative  0 0  Alanine transaminase (ALT) IU/L 13.8~627 34~673  1.0 Mean ALT level  190.153 187.393 SD  161.231 177.618  Aspartate aminotransferase (AST) IU/L 26.5~345 28~556  0.9999 Mean AST  96.74 142.427 SD  71.699 153.539  Serum total bilirubin (STB) umol/L 10.1~48.63 6.5~109.5  1.0 Mean STB  19.208 20.643 SD  8.926 18.127  Conjugated bilirubin (CB) umol/L 2.4~16.4 2.3~99.2  1.0 Mean CB  6.923 9.408 SD  3.542 17.208  Unconjugated bilirubin (UCB) umol/L 6~34.13 3.3~29.3  1.0 Mean UCB  12.284 11.182 SD  5.951 4.921  HBV-DNA IU/mL 5.12 E + 03~1.12E+08 6.34 E + 04~9.40E+08  0.0096 Mean HBV-DNA  2.521 E + 07 5.655 E + 07 SD  3.221 E + 07 1.717 E + 08


### 3.2. Protein Identification and Quantification by iTRAQ

Next, we quantified the serum proteins in these LSSD-CHB and DHSM-CHB patients using iTRAQ. Initially, a total of 371,034 spectra were generated by liquid chromatography coupled to mass spectrometry (LC-MS/MS) analysis. Of them, 98,243 spectra (5,591 unique peptides) were aligned to 842 proteins from 666 families. The mass distribution of identified proteins (Figure1(a)) suggested by Mascot revealed 170 (98.69%) were above 10 kDa, of which 170 (20.19%) and 141 (16.75%) were 10 to 20 kDa and above 100 kDa, respectively. We also counted the proteins aligned with significant peptides, shown in Figure 1(b), and 547 (64.96%) proteins were aligned by two and more peptides. In addition, the distribution of protein sequence coverage is shown in Figure 1(c). Protein sequence coverage with 40~100%, 30~40%, 20~30%, 10~20%, and under 10% variation accounted for 8.79%, 14.25%, 17.70%, 23.28%, and 35.99%, respectively. In Figure 1(d), we showed correlation between two samples and found LSSD-CHB samples were closer to DHSM-CHB samples than HCTL.Figure 1
Identification and analysis of serum proteome of CHB infected patients. (a) Distribution of protein mass of identified proteins. (b) Number of peptides that match to proteins as indicated by MASCOT 2.4.0. (c) Coverage of identified proteins in CHB patient serum. (d) Correlation between samples calculated by corrplot.
(a) (b) (c) (d)


### 3.3. Identification of Differentially Expressed Proteins

Differentially expressed proteins were defined as those showed greater than 1.2-fold change in relative abundance and ap value < 0.05. Compared to HCTL we identified a total of 392 proteins differentially expressed (Table S2), of which 273 were identified in LSSD-CHB group and 345 in DHSM-CHB group. As shown in the volcano plots, we identified 172 upregulated and 101 downregulated proteins in LSSD-CHB group (Figure 2(a)) and 199 upregulated and 146 downregulated proteins in DHSM-CHB group (Figure 2(b)), compared to HCTL group. Venn diagram (Figure 2(c)) revealed LSSD-CHB and DHSM-CHB shared 142 upregulated and 84 downregulated proteins; 30 and 57 proteins were exclusively upregulated in LSSD-CHB and DHSM-CHB, respectively; 17 and 62 proteins were exclusively downregulated in LSSD-CHB and DHSM-CHB, respectively; and no protein was identified with upregulation in one CHB subtype but with downregulation in another.Figure 2
Identification of differentially expressed proteins in LSSD-CHB and DHSM-CHB patients compared with HCTL. (a) Volcano plot of differentially expressed proteins between LSSD-CHB and HCTL groups. (b) Volcano plot of differentially expressed proteins between DHSM-CHB and HCTL groups. (c) Venn diagram of up- and downregulated proteins in LSSD-CHB and DHSM-CHB groups.
(a) (b) (c)


### 3.4. Potential Biomarkers for CHB

The identification of proteins differentially expressed in LSSD-CHB and DHSM-CHB groups relative to the HCTL group was of interest as these could provide leads for potentially useful diagnostic and prognostic biomarkers. First, we examined those 142 commonly upregulated and 84 commonly downregulated proteins. As shown in Table2, the largest upregulated protein family was immunoglobulin related protein, showing 20 upregulated and 3 downregulated proteins identified. In clinical immunology, levels of immunoglobulins especially IgG can be used to characterize viral hepatitis in patients [28, 29]. Four IgG subclasses (IgG1 to IgG4) differ in their heavy chain constant regions and have different effects on virus-cell fusion inhibition, virus neutralization, and overall course of infection, as have been reported for various viruses including HIV [30] and HBV [31]. Highly expressed proteins encoding heavy chains for immunoglobulins including IGHG1, IGHG3, IGHG4, and IGH@ have been reported with upregulation in HBV [32, 33] and HCC patients [34]. Other upregulated protein families such as heat shock protein, histone, ras-related protein, and von Willebrand factor identified in current study have also been reported in patients infected by HBV or hepatitis C virus (HCV) [35–38]. The largest downregulated protein family was complement, 15 complement proteins downregulated in LSSD-CHB with 0.82- to 0.37-fold change and in DHSM-CHB with 0.77- to 0.31-fold change. Other protein families like insulin-like growth factor binding protein and serum amyloid protein were also decreased in CHB patients in comparison to HCTL group. In addition, several known upregulated proteins from other families in patients infected by HBV or HCV (Table 2), such as apolipoproteins (APOA2, APOB, and APOB-variant) [39, 40], A2M (alpha-2-macroglobulin) [41], alpha-actinin-3 (ACTN3) [42, 43], vimentin (VIM) [38], and putative uncharacterized proteins (DKFZp686N02209 and DKFZp686I04196) [34, 44, 45], were identified in LSSD-CHB and DHSM-CHB groups. The different expression of proteins in the serum of CHB patients indicates they may have functions in response of HBV and CHB processing and can be used as biomarkers in clinical diagnosis.Table 2
Commonly up- and downregulated proteins in LSSD-CHB and SSDHS-CHB groups (part).
Family UniProt_ID Gene_name Description Number of significant matches Number of significant sequences emPAI LSSD-CHB versus HCTL SSDHS-CHB versus HCTL F C a p   v a l u e b F C a p   v a l u e b Immunoglobulin related proteins A0A087WV47_HUMAN IGHG1 Ig gamma-1 chain C region 1541 14 5.37 1.84 6.30 E - 05 1.47 2.71 E - 04 A0A087WYC5_HUMAN IGHG1 Ig gamma-1 chain C region 1501 14 4.82 1.87 3.51 E - 05 1.48 1.69 E - 04 A0A087X1C7_HUMAN IGHG1 Ig gamma-1 chain C region 1511 13 4.79 1.84 6.12 E - 05 1.47 2.60 E - 04 A0A087WXL8_HUMAN IGHG3 Ig gamma-3 chain C region 739 11 2.07 1.84 5.45 E - 05 1.47 1.12 E - 04 A0A0G2JPD4_HUMAN IGHG4 Ig gamma-4 chain C region (fragment) 31 6 1.56 1.69 6.47 E - 04 1.24 4.12 E - 02 KV112_HUMAN  Ig kappa chain V-I region Kue 2 2 0.53 1.82 1.39 E - 03 1.51 9.53 E - 04 KV311_HUMAN  Ig kappa chain V-III region IARC/BL41 2 1 0.49 1.61 5.86 E - 03 1.28 1.64 E - 02 LV302_HUMAN  Ig lambda chain V-III region LOI 25 3 4.01 1.73 1.75 E - 04 1.35 8.79 E - 04 LV301_HUMAN  Ig lambda chain V-III region SH 6 1 0.61 1.99 1.59 E - 07 1.56 5.32 E - 07 S6B2B6_HUMAN  IgG H chain 384 3 0.6 2.05 1.06 E - 04 1.57 9.46 E - 04 S6BAM6_HUMAN  IgG H chain 389 4 0.9 2.05 4.60 E - 05 1.56 5.55 E - 04 S6BGE0_HUMAN  IgG H chain 394 4 1.13 2.04 4.84 E - 05 1.56 6.39 E - 04 S6C4S4_HUMAN  IgG H chain 384 3 0.73 2.05 9.63 E - 05 1.57 5.91 E - 04 S6C4R7_HUMAN  IgG L chain 257 6 6.68 1.53 2.61 E - 02 1.27 4.54 E - 02 Q6GMX6_HUMAN IGH@ IGH@ protein 1524 14 6.04 1.83 6.27 E - 05 1.46 3.15 E - 04 A0A0F7TC28_HUMAN IGHV4-4 IGHV4-4 protein (fragment) 3 2 1.27 1.49 9.67 E - 03 1.24 2.27 E - 02 Q5FWF9_HUMAN IGL@ IGL@ protein 143 6 6.03 1.60 1.35 E - 02 1.32 2.83 E - 02 Q6PIK1_HUMAN IGL@ IGL@ protein 197 5 4.45 1.59 1.37 E - 02 1.29 4.62 E - 02 Q9UL84_HUMAN  Myosin-reactive immunoglobulin heavy chain variable region (fragment) 6 3 1.69 1.52 1.58 E - 02 1.46 2.04 E - 04 Q9UL79_HUMAN  Myosin-reactive immunoglobulin light chain variable region (fragment) 2 1 0.57 0.65 6.56 E - 04 0.50 1.93 E - 12 A0A0C4DH33_HUMAN IGHV1-24 Protein IGHV1-24 (fragment) 5 2 0.84 0.82 5.43 E - 03 0.81 1.38 E - 02 A0A0A0MT74_HUMAN IGKV1-16 Protein IGKV1-16 (fragment) 5 1 0.9 1.58 3.55 E - 03 1.26 3.22 E - 02 A0A075B6I9_HUMAN IGLV7-46 Protein IGLV7-46 (fragment) 31 3 1.41 1.54 9.85 E - 03 1.36 2.46 E - 03  Complement B7Z1F8_HUMAN  cDNA FLJ53025, highly similar to complement C4-B 2230 12 56.68 0.78 1.27 E - 03 0.77 2.33 E - 03 B4E3S6_HUMAN  cDNA FLJ58413, highly similar to complement component C7 63 10 1.74 0.55 8.69 E - 10 0.50 8.03 E - 15 A8K2T4_HUMAN  cDNA FLJ78207, highly similar to human complement protein component C7 mRNA 86 15 1.31 0.55 1.62 E - 09 0.50 2.39 E - 13 B2RA39_HUMAN  cDNA, FLJ94686, highly similar toHomo sapiens complement factor H-related 5 (CFHL5), mRNA 35 5 0.51 0.54 7.03 E - 12 0.57 1.44 E - 11 CO4A_HUMAN C4A Complement C4-A 5889 65 21.28 0.79 2.87 E - 03 0.75 7.70 E - 04 CO4B_HUMAN C4B Complement C4-B 5983 66 23.03 0.79 2.95 E - 03 0.74 5.91 E - 04 A0A024R035_HUMAN C9 Complement component 9, isoform CRA_a 225 17 5.12 0.67 1.36 E - 05 0.54 6.61 E - 13 CO6_HUMAN C6 Complement component C6 92 12 0.83 0.50 3.34 E - 12 0.48 4.06 E - 15 CO8A_HUMAN C8A Complement component C8 alpha chain 66 8 1.51 0.66 1.94 E - 06 0.53 8.36 E - 15 F5GY80_HUMAN C8B Complement component C8 beta chain 133 16 3.28 0.65 6.59 E - 07 0.61 1.07 E - 09 CO8G_HUMAN C8G Complement component C8 gamma chain 41 8 6.86 0.61 8.80 E - 08 0.50 3.38 E - 14 B1AKG0_HUMAN CFHR1 Complement factor H-related protein 1 216 6 4.26 0.66 1.30 E - 06 0.75 4.80 E - 04 FHR3_HUMAN CFHR3 Complement factor H-related protein 3 45 6 1.19 0.82 7.05 E - 03 0.77 2.36 E - 03 FHR4_HUMAN CFHR4 Complement factor H-related protein 4 31 3 0.41 0.37 3.90 E - 23 0.31 1.62 E - 44 A0A0S2Z4I5_HUMAN CFP Complement factor properdin isoform 1 (fragment) 13 2 0.17 0.58 2.21 E - 11 0.72 2.90 E - 05  Apolipoprotein APOA2_HUMAN APOA2 Apolipoprotein A-II 421 2 1.84 1.48 4.64 E - 02 1.26 3.72 E - 02 A0A0B4RUS7_HUMAN APOA5 Apolipoprotein A-V, isoform CRA_a 15 7 1 0.73 2.03 E - 05 0.62 2.95 E - 08 E1B4S9_HUMAN APOB Apolipoprotein B (fragment) 129 7 10.94 1.97 1.23 E - 05 1.53 9.70 E - 05 C0JYY2_HUMAN APOB Apolipoprotein B (including Ag(X) antigen) 5227 215 21.89 1.84 2.08 E - 04 1.44 1.07 E - 03 Q59HB3_HUMAN  Apolipoprotein B variant (fragment) 1661 69 16.33 1.85 2.79 E - 04 1.41 1.40 E - 03 B0YIW2_HUMAN APOC3 Apolipoprotein C-III 577 5 8.48 0.66 1.44 E - 05 0.55 3.02 E - 09 APOC4_HUMAN APOC4 Apolipoprotein C-IV 26 5 3.84 0.80 1.35 E - 03 0.60 8.22 E - 09  Histone H15_HUMAN HIST1H1B Histone H1.5 4 2 0.35 2.02 8.32 E - 06 1.72 1.02 E - 04 A0A024R017_HUMAN HIST1H2AC Histone H2A 21 4 2.27 4.67 1.78 E - 28 3.63 4.54 E - 33 C9J0D1_HUMAN H2AFV Histone H2A 13 3 1.11 4.84 3.09 E - 29 3.73 2.99 E - 37 A0A024QZZ7_HUMAN HIST1H2BD Histone H2B 12 4 1.15 4.58 1.39 E - 29 3.38 6.00 E - 31 B2R4P9_HUMAN H3F3A Histone H3 9 3 0.88 4.98 5.44 E - 32 3.72 9.13 E - 39 Q5TEC6_HUMAN HIST2H3PS2 Histone H3 4 3 0.6 2.59 1.10 E - 08 1.87 3.39 E - 06 B2R4R0_HUMAN HIST1H4J Histone H4 39 5 13.27 4.47 3.83 E - 35 3.41 3.84 E - 45  Heat shock protein A0A024RD80_HUMAN HSP90AB1 Heat shock protein 90 kDa alpha (cytosolic), class B member 1, isoform CRA_a 12 6 0.31 1.46 3.59 E - 02 1.51 2.77 E - 05 HS90A_HUMAN HSP90AA1 Heat shock protein HSP 90-alpha 28 10 0.55 1.58 5.45 E - 03 1.65 3.81 E - 07  Insulin-like growth factor binding protein C1K3N3_HUMAN IGFBP1 Insulin-like growth factor binding protein 1 (fragment) 3 2 0.22 0.61 2.62 E - 07 0.60 5.40 E - 09 A0A024R1U8_HUMAN IGFBP4 Insulin-like growth factor binding protein 4, isoform CRA_a 2 1 0.1 0.66 8.52 E - 06 0.66 1.00 E - 06 A0A024R433_HUMAN IGFBP5 Insulin-like growth factor binding protein 5, isoform CRA_a 3 2 0.18 0.83 3.37 E - 03 0.80 4.98 E - 02  Ras-related protein A0A024R7I3_HUMAN RAB8A RAB8A, member RAS oncogene family, isoform CRA_a 11 4 1.08 1.75 1.87 E - 03 1.73 9.62 E - 06 A0A024RB87_HUMAN RAP1B RAP1B, member of RAS oncogene family, isoform CRA_a 10 4 1.66 1.85 8.55 E - 05 1.96 7.63 E - 11 RAB10_HUMAN RAB10 Ras-related protein Rab-10 9 3 0.71 1.74 2.50 E - 03 1.72 2.60 E - 05 RB11B_HUMAN RAB11B Ras-related protein Rab-11B 5 4 0.73 1.91 3.03 E - 05 1.57 1.03 E - 05 RAB1B_HUMAN RAB1B Ras-related protein Rab-1B 12 6 1.5 1.73 2.07 E - 03 1.73 7.38 E - 06 RB27B_HUMAN RAB27B Ras-related protein Rab-27B 5 4 0.7 1.70 1.33 E - 03 1.59 2.26 E - 05 RAB7A_HUMAN RAB7A Ras-related protein Rab-7a 16 6 1.94 1.60 2.16 E - 02 1.66 1.48 E - 04  Serum amyloid D3DQX7_HUMAN SAA1 Serum amyloid A protein 59 5 9.88 0.53 2.58 E - 13 0.40 3.36 E - 24 SAA1_HUMAN SAA1 Serum amyloid A-1 protein 40 4 4.99 0.48 1.21 E - 15 0.38 4.55 E - 28 SAA2_HUMAN SAA2 Serum amyloid A-2 protein 24 4 4.99 0.48 7.27 E - 16 0.36 3.08 E - 31 SAMP_HUMAN APCS Serum amyloid P-component 228 8 15.23 0.83 8.13 E - 03 0.70 4.45 E - 05  von Willebrand factor L8E853_HUMAN VWF von Willebrand factor 189 38 0.98 1.68 2.69 E - 03 1.41 9.67 E - 04 VWF_HUMAN VWF von Willebrand factor 186 38 0.93 1.67 3.05 E - 03 1.41 7.94 E - 04 F o l d   a change provided by MASCOT. p   b values calculated by edgeR to show the significance of different expression.We next analyzed the potential functions of commonly differentially expressed serum proteins in LSSD-CHB and DHSM-CHB groups using DAVID Bioinformatics Resources 6.7 [24] and STRING v10 [25]. Cellular component annotation (Figure 3(a)) showed 63 and 7 proteins were “extracellular region” (GO: 0005576, GO: 0005615, and GO: 0044421) and “lipids” (GO: 0032994 and GO: 0034358), respectively. However biological process annotation (Figure 3(b)) showed most of the differentially expressed proteins associated with immune response, including “acute inflammatory response” (GO: 0002526), “response to wounding” (GO: 0009611), “inflammatory response” (GO: 0006954), “complement activation” (GO: 0006956), “defense response” (GO: 0006952), “humoral immune response mediated by circulating immunoglobulin” (GO: 0002455), “immune effector process” (GO: 0002252), “B cell mediated immunity” (GO: 0019724), and “activation of immune response” (GO: 0002253). It has been well studied that immunological events are necessary to control hepatitis B virus (HBV) infection [46, 47]. In addition, KEGG pathway analysis also showed differentially expression proteins function mainly in the pathways of “complement and coagulation cascades” (hsa04610), “systemic lupus erythematosus” (hsa05322), “focal adhesion” (hsa04510), and “viral carcinogenesis” (hsa05203). Overall, differentially expressed proteins in both LSSD-CHB and DHSM-CHB groups have potential ability to be used as biomarkers.Figure 3
Gene Ontology annotation for shared differentially expressed proteins in LSSD- and DHSM-CHB groups: (a) cellular component, (b) biological process.
(a) (b)


### 3.5. Dysregulated Proteins Detected Exclusively in LSSD-CHB and DHSM-CHB

Next, we examined differentially expressed proteins exclusively in LSSD-CHB and DHSM-CHB groups. A total of 30 upregulated and 17 downregulated proteins were specifically identified in LSSD-CHB patient serum samples (Table3). Among them 11 upregulated immunoglobulin related proteins, gelsolin (GSN), serum paraoxonase/lactonase 3 (PON3), likely SNC73 protein, transient receptor potential cation channel subfamily M member 8 (TRPM8), and several uncharacterized proteins (DKFZp686M08189, DKFZp686C02220, and DKFZp686K04218) attracted our attention due to their high abundance. Serum PON3 concentrations have been reported to increase in patients with CHB or cirrhosis and showed significant direct correlations with the degree of periportal abnormalities including fibrosis and with serum FAS (a marker of antiapoptosis) concentrations [48]; however, serum gelsolin level has been reported to reduce significantly in patients with acute liver failure (47%), myocardial infarction (69%), sepsis (51%), and myonecrosis (66%) [49]. Among the specifically downregulated serum proteins in LSSD-CHB patients fibulin-1 (FBLN1) is a tumor suppressor in hepatocellular carcinoma [50]. Proteins specifically differentially expressed in LSSD-CHB patients were predicted to function mainly in biological processes of “protein activation cascade” (GO: 0072376), “regulation of response to wounding” (GO: 1903034), “blood coagulation, fibrin clot formation” (GO: 0072378), “negative regulation of response to stimulus” (GO: 0048585), and “acute-phase response” (GO: 0006953).Table 3
Up- and downregulated serum proteins specifically in LSSD-CHB.
UniProt_ID Gene_name Description Number of significant matches Number of significant sequences emPAI F C a p   v a l u e b Upregulated A0A024R6I9_HUMAN SERPINA4 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 4, isoform CRA_a 79 12 3.79 1.78 5.24 E - 05 A0A075B6K3_HUMAN IGLV2-11 Protein IGLV2-11 (fragment) 8 1 0.9 1.43 4.94 E - 02 A0A075B6K6_HUMAN IGLV4-3 Protein IGLV4-3 4 1 0.51 1.54 1.23 E - 02 A0A075B6N7_HUMAN IGHA2 Ig alpha-2 chain C region (fragment) 80 6 1.94 1.44 4.71 E - 02 A0A0A0MS51_HUMAN GSN Gelsolin 146 16 2.42 1.51 2.86 E - 02 A0A0B4J1V4_HUMAN IGHV1-46 Protein IGHV1-46 (fragment) 2 1 0.23 1.44 4.22 E - 02 A0N7J6_HUMAN  REV25-2 (fragment) 2 2 0.47 1.92 3.69 E - 03 A0N8J1_HUMAN  V(k)3 sequence of NG9 gene from fetal liver DNA (fragment) 6 1 1.1 1.49 1.49 E - 02 CPN2_HUMAN CPN2 Carboxypeptidase N subunit 2 94 11 2.09 1.48 1.88 E - 02 D3DNU8_HUMAN KNG1 Kininogen 1, isoform CRA_a 91 14 3.3 1.50 1.14 E - 02 FETUA_HUMAN AHSG Alpha-2-HS-glycoprotein 57 3 0.64 1.70 1.36 E - 03 FETUB_HUMAN FETUB Fetuin-B 9 3 0.29 1.65 6.32 E - 04 HV208_HUMAN  Ig heavy chain V-II region SESS 2 1 0.21 2.27 1.25 E - 08 ITB1_HUMAN ITGB1 Integrin beta-1 3 2 0.06 1.46 3.43 E - 02 KV119_HUMAN  Ig kappa chain V-I region Wes 21 2 2.16 1.52 6.37 E - 03 KV308_HUMAN  Ig kappa chain V-III region CLL 4 1 0.49 1.65 8.16 E - 03 LV204_HUMAN  Ig lambda chain V-II region TRO 7 2 0.96 1.59 1.25 E - 02 PCYOX_HUMAN PCYOX1 Prenylcysteine oxidase 1 48 9 1.89 1.43 3.70 E - 02 PEDF_HUMAN SERPINF1 Pigment epithelium-derived factor 23 9 1.54 1.48 1.56 E - 02 PON3_HUMAN PON3 Serum paraoxonase/lactonase 3 179 12 3.89 1.51 4.11 E - 02 Q6MZX9_HUMAN DKFZp686M08189 Putative uncharacterized protein DKFZp686M08189 124 7 1.41 1.43 3.95 E - 02 Q6N091_HUMAN DKFZp686C02220 Putative uncharacterized protein DKFZp686C02220 (fragment) 71 6 1.2 1.46 2.31 E - 02 Q6ZVX0_HUMAN  cDNA FLJ41981 fis, clone SMINT2011888, highly similar to protein Tro alpha1 H,myeloma 125 7 1.38 1.43 4.13 E - 02 Q7Z379_HUMAN DKFZp686K04218 Putative uncharacterized protein DKFZp686K04218 (fragment) 124 7 1.44 1.43 3.96 E - 02 Q8TE63_HUMAN  Immunoglobulin light chain variable region (fragment) 4 2 0.97 1.45 4.51 E - 02 Q96K68_HUMAN  cDNA FLJ14473 fis, clone MAMMA1001080, highly similar toHomo sapiens SNC73 protein (SNC73) mRNA 205 9 1.77 1.45 2.93 E - 02 Q9NPP6_HUMAN  Immunoglobulin heavy chain variant (fragment) 125 7 1.75 1.43 4.92 E - 02 Q9UL83_HUMAN  Myosin-reactive immunoglobulin light chain variable region (fragment) 14 2 2.98 1.54 5.43 E - 03 Q9UL89_HUMAN  Myosin-reactive immunoglobulin heavy chain variable region (fragment) 18 5 8.63 1.49 2.16 E - 02 TRPM8_HUMAN TRPM8 Transient receptor potential cation channel subfamily M member 8 3 2 0.04 1.60 6.39 E - 03  Downregulated A0A024QZK7_HUMAN HK1 Hexokinase 5 1.00 E + 00 0.03 0.82 7.31 E - 03 A0A024R2X3_HUMAN HYAL1 Hyaluronidase 12 5.00 E + 00 0.55 0.73 2.82 E - 05 A0A024R451_HUMAN SERPINE2 Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2, isoform CRA_a 17 8 1.05 0.83 2.36 E - 03 A0A024RAG6_HUMAN C1QA Complement component 1, q subcomponent, A chain, isoform CRA_a 12 4 1.08 0.75 4.11 E - 04 A0A0S2Z3Y1_HUMAN LGALS3BP Lectin galactoside-binding soluble 3 binding protein isoform 1 (fragment) 247 14 4.17 0.71 2.49 E - 06 A0A0S2Z4D4_HUMAN PLP1 Proteolipid protein 1 isoform 1 (fragment) 2 1 0.11 0.40 1.65 E - 04 ADT4_HUMAN SLC25A31 ADP/ATP translocase 4 5 1 0.16 0.65 1.28 E - 02 B1AHL2_HUMAN FBLN1 Fibulin-1 74 8 0.92 0.78 4.33 E - 04 CALU_HUMAN CALU Calumenin 12 7 0.86 0.76 2.45 E - 04 CFAH_HUMAN CFH Complement factor H 689 31 4.02 0.83 8.87 E - 03 LBP_HUMAN LBP Lipopolysaccharide-binding protein 148 11 3.18 0.79 3.74 E - 04 LRP1_HUMAN LRP1 Prolow-density lipoprotein receptor-related protein 1 37 20 0.16 0.83 7.44 E - 03 LTBP1_HUMAN LTBP1 Latent-transforming growth factor beta-binding protein 1 19 8 0.16 0.77 8.36 E - 04 NUCB1_HUMAN NUCB1 Nucleobindin-1 17 7 0.66 0.78 5.80 E - 04 Q8NBH6_HUMAN  Fibulin-1 125 11 2.25 0.75 1.26 E - 04 Q9HCC1_HUMAN  Single chain Fv (fragment) 10 2 0.96 0.79 4.29 E - 04 THRB_HUMAN F2 Prothrombin 1145 18 6.65 0.78 6.78 E - 04 F o l d   a change provided by MASCOT. p   b values calculated by edgeR to show the significance of different expression.We also identified 57 upregulated and 62 downregulated proteins exclusively in DHSM-CHB patients (Table4). Two IGL@ proteins (Q6GMX4_HUMAN and Q6PIQ7_HUMAN) were specifically upregulated in DHSM-CHB patients with 1.27-fold change. Transthyretin (TTR), upregulated 1.34-fold in DHSM-CHB, can be induced by hepatitis C virus and activate TGF-β signaling pathway with furin [51]. Interestingly, we found three members of tubulin (TUBA4A, TUBB1, and TUBB8) were upregulated only in DHSM-CHB patients compared with HCTL. Although there are few reports about tubulin and HBV, it is well known that 42 kDa tubulin alpha-6 chain fragment in well-differentiated hepatocellular carcinoma tissues is from patients infected with HCV [52]. In addition, we found actinin, alpha 1 (ACTN1), which can directly interact with HCV [53], GAPDH, which can bind to the HBV posttranscriptional regulatory element [54], and polymeric immunoglobulin receptor (PIGR), the main transporter of IgA [55], were upregulated in DHSM-CHB but not in LSSD-CHB. Among DHSM-CHB specifically downregulated proteins we identified three members of keratin type I (KRT9, KRT10, and KRT14) and another three members of keratin type II (KRT1, KRT2, and KRT6B). Although there is no evidence showing relation between these six keratin proteins with CHB or other liver diseases, variant keratins are associated with progression of fibrosis during chronic hepatitis C infection [56]. Differentially expressed proteins exclusively detected in DHSM-CHB patients were predicted to be involved in the biological processes of “immune system process” (GO: 0002376), “response to stress” (GO: 0006950), “defense response” (GO: 0006952), “immune response” (GO: 0006955), and “single-organism metabolic process” (GO: 0044710).Table 4
Up- and downregulated serum proteins exclusively in DHSM-CHB.
UniProt_ID Gene_name Description Number of significant matches Number of significant sequences emPAI F C a p   v a l u e b Upregulated A0A024R145_HUMAN ALDOB Fructose-bisphosphate aldolase 5 3 0.22 1.29 3.81 E - 02 A0A024R1N1_HUMAN MYH9 Myosin, heavy polypeptide 9, nonmuscle, isoform CRA_a 17 9 0.13 1.43 1.34 E - 03 A0A024R5H8_HUMAN RAB6A RAB6A, member RAS oncogene family, isoform CRA_b 8 3 0.57 1.43 4.84 E - 03 A0A024R694_HUMAN ACTN1 Actinin, alpha 1, isoform CRA_a 20 10 0.42 1.33 1.97 E - 02 A0A024R6G3_HUMAN FBLN5 Fibulin 5, isoform CRA_b 6 3 0.27 1.33 6.31 E - 03 A0A024R9T1_HUMAN hCG_39634 HCG39634, isoform CRA_a 2 2 0.26 1.49 4.90 E - 03 A0A024RDB8_HUMAN HPSE Heparanase, isoform CRA_a 23 6 0.55 1.36 1.03 E - 03 A0A024RDL8_HUMAN ASL Argininosuccinate lyase isoform 1 10 5 0.39 1.65 2.02 E - 07 A0A087WT59_HUMAN TTR Transthyretin 175 7 5.66 1.34 3.42 E - 02 A0A087WUL0_HUMAN TKFC Triokinase/FMN cyclase 3 3 0.15 1.28 9.35 E - 03 A0A0A0MSD0_HUMAN SVEP1 Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 4 3 0.02 1.36 7.51 E - 03 A0A0A0MT32_HUMAN LIPA Lysosomal acid lipase/cholesteryl ester hydrolase 2 1 0.09 1.38 4.78 E - 03 A0A0C4DFP6_HUMAN CRTAC1 Cartilage acidic protein 1 6 5 0.24 1.23 3.57 E - 02 A0A0S2Z3F6_HUMAN CETP Cholesteryl ester transfer protein plasma isoform 1 (fragment) 95 14 3.56 1.36 1.42 E - 03 A0A125QYY9_HUMAN  IBM-B2 heavy chain variable region (fragment) 12 2 0.83 1.42 2.25 E - 03 A0N7I9_HUMAN F5-20 F5-20 (fragment) 4 1 0.47 1.68 2.31 E - 03 A2NYU9_HUMAN  Heavy chain Fab (fragment) 2 1 0.48 1.29 3.92 E - 02 A8K486_HUMAN  Peptidyl-prolyl cis-trans isomerase 2 2 0.32 1.40 4.10 E - 02 ADIPO_HUMAN ADIPOQ Adiponectin 27 5 2.13 1.32 1.12 E - 02 APOF_HUMAN APOF Apolipoprotein F 7 3 0.5 1.29 1.84 E - 02 ASSY_HUMAN ASS1 Argininosuccinate synthase 4 3 0.18 1.49 1.09 E - 04 ATPB_HUMAN ATP5B ATP synthase subunit beta, mitochondrial 11 2 0.22 1.34 3.74 E - 02 B3KTV0_HUMAN  cDNA FLJ38781 fis, clone LIVER2000216, highly similar to HEAT SHOCK COGNATE 71 kDa PROTEIN 5 5 0.21 1.29 3.41 E - 02 B4DQK4_HUMAN  cDNA FLJ53743, highly similar to tubulin alpha-3 chain 21 6 0.81 1.61 1.06 E - 03 B4DVA7_HUMAN  Beta-hexosaminidase 3 2 0.15 1.26 1.84 E - 02 B7Z478_HUMAN PSMB2 Proteasome (prosome, macropain) subunit, beta type, 2, isoform CRA_b 2 1 0.3 1.65 1.96 E - 03 BPIB1_HUMAN BPIFB1 BPI fold-containing family B member 1 11 6 0.51 1.28 1.34 E - 02 BTD_HUMAN BTD Biotinidase 47 6 1.12 1.54 1.69 E - 06 CAMP_HUMAN CAMP Cathelicidin antimicrobial peptide 2 1 0.29 1.43 2.64 E - 03 CAND1_HUMAN CAND1 Cullin-associated NEDD8-dissociated protein 1 2 1 0.02 1.36 2.60 E - 03 CATD_HUMAN CTSD Cathepsin D 2 1 0.13 1.28 3.06 E - 02 DHSO_HUMAN SORD Sorbitol dehydrogenase 4 3 0.32 1.67 2.46 E - 07 FIBA_HUMAN FGA Fibrinogen alpha chain 102 22 2.36 1.36 3.18 E - 03 G3P_HUMAN GAPDH Glyceraldehyde-3-phosphate dehydrogenase 19 7 1.37 1.37 5.07 E - 03 G3V5Z7_HUMAN PSMA6 Proteasome subunit alpha type 7 4 0.58 1.44 9.01 E - 04 HV310_HUMAN  Ig heavy chain V-III region HIL 3 1 0.5 1.48 1.12 E - 03 HV320_HUMAN  Ig heavy chain V-III region GAL 36 3 1.91 1.32 2.19 E - 02 KV404_HUMAN  Ig kappa chain V-IV region B17 34 2 1.45 1.45 4.98 E - 04 LDHB_HUMAN LDHB L-lactate dehydrogenase B chain 4 2 0.33 1.65 2.13 E - 06 M0QZB5_HUMAN PPFIA4 Liprin-alpha-4 (fragment) 2 1 0.03 1.33 2.70 E - 02 MARCO_HUMAN MARCO Macrophage receptor MARCO 2 1 0.11 1.31 2.89 E - 02 MDR3_HUMAN ABCB4 Phosphatidylcholine translocator ABCB4 2 1 0.04 1.32 1.23 E - 02 OTUB1_HUMAN OTUB1 Ubiquitin thioesterase OTUB1 2 1 0.09 1.27 2.92 E - 02 PIGR_HUMAN PIGR Polymeric immunoglobulin receptor 7 4 0.18 1.34 4.27 E - 03 PSA3_HUMAN PSMA3 Proteasome subunit alpha type-3 8 4 0.58 1.31 4.84 E - 02 PSB1_HUMAN PSMB1 Proteasome subunit beta type-1 4 3 0.5 1.47 1.25 E - 03 Q6GMX4_HUMAN IGL@ IGL@ protein 442 6 7.15 1.27 3.98 E - 02 Q6PIQ7_HUMAN IGL@ IGL@ protein 441 6 6.39 1.27 4.86 E - 02 Q6ZNX5_HUMAN  CDNA FLJ26936 fis, clone RCT06808 5 1 0.39 1.78 2.10 E - 08 Q9UL82_HUMAN  Myosin-reactive immunoglobulin light chain variable region (fragment) 46 3 2.11 1.41 1.22 E - 03 QSOX1_HUMAN QSOX1 Sulfhydryl oxidase 1 97 20 2.33 1.24 4.15 E - 02 TBA4A_HUMAN TUBA4A Tubulin alpha-4A chain 24 8 0.86 1.55 1.38 E - 03 TBB1_HUMAN TUBB1 Tubulin beta-1 chain 6 4 0.33 1.30 2.87 E - 02 TBB8_HUMAN TUBB8 Tubulin beta-8 chain 7 5 0.42 1.32 3.10 E - 02 TRML1_HUMAN TREML1 Trem-like transcript 1 protein 3 1 0.09 1.42 1.05 E - 03 TSP1_HUMAN THBS1 Thrombospondin-1 365 34 4.23 1.37 1.66 E - 03 TYPH_HUMAN TYMP Thymidine phosphorylase 3 3 0.19 1.23 4.67 E - 02  Downregulated A0A024CIM4_HUMAN  Carboxylic ester hydrolase 7 3 0.17 0.81 1.43 E - 02 A0A024R6I6_HUMAN SERPINA10 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 10, isoform CRA_b 65 12 2.73 0.75 1.46 E - 04 A0A024R6K8_HUMAN WARS Tryptophanyl-tRNA synthetase, isoform CRA_a 5 2 0.1 0.82 4.87 E - 02 A0A024R853_HUMAN IQCE IQ motif containing E, isoform CRA_b 2 1 0.04 0.74 1.67 E - 03 A0A024R9J3_HUMAN COLEC10 Collectin subfamily member 10 (C-type lectin), isoform CRA_a 4 2 0.28 0.83 2.40 E - 02 A0A075B6S2_HUMAN IGKV2D-29 Protein IGKV2D-29 (fragment) 26 3 2.47 0.77 2.05 E - 03 A0A087X054_HUMAN HYOU1 Hypoxia upregulated protein 1 4 2 0.08 0.58 2.93 E - 08 A0A0A7C3P2_HUMAN HLA-A MHC class I antigen (fragment) 8 3 0.72 0.77 1.24 E - 03 A0A0J9YX35_HUMAN  Uncharacterized protein (fragment) 2 1 0.51 0.74 3.43 E - 03 A0A0X9TD47_HUMAN  MS-D1 light chain variable region (fragment) 30 3 5.49 0.83 1.31 E - 02 A2AP_HUMAN SERPINF2 Alpha-2-antiplasmin 67 10 1.86 0.74 2.85 E - 04 A2J1N4_HUMAN  Rheumatoid factor RF-IP24 (fragment) 10 2 1.08 0.80 4.59 E - 03 A2J1N5_HUMAN  Rheumatoid factor RF-ET6 (fragment) 15 1 1.65 0.79 6.40 E - 04 A2MYD0_HUMAN V1-17 V1-17 protein (fragment) 8 3 1.87 0.83 1.21 E - 02 A2NB45_HUMAN  Cold agglutinin FS-1 L-chain (fragment) 23 2 1.39 0.69 3.83 E - 04 A3RKG7_HUMAN  Coagulation factor VII (fragment) 17 6 2.02 0.77 7.55 E - 04 ALBU_HUMAN ALB Serum albumin 1841 31 17.82 0.67 1.64 E - 06 AMBP_HUMAN AMBP Protein AMBP 34 3 0.75 0.76 4.96 E - 04 ATS13_HUMAN ADAMTS13 A disintegrin and metalloproteinase with thrombospondin motifs 13 22 8 0.26 0.81 6.52 E - 03 B2RBZ5_HUMAN  cDNA, FLJ95778, highly similar toHomo sapiens serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 10 (SERPINA10), mRNA 58 11 2.2 0.76 1.77 E - 04 B4DL32_HUMAN  cDNA FLJ59922, highly similar to keratin, type II cytoskeletal 5 7 2 0.33 0.83 2.87 E - 02 B4DN21_HUMAN  cDNA FLJ53365, highly similar toHomo sapiens fibronectin 1 (FN1), transcript variant 4, mRNA 384 8 6.85 0.77 1.76 E - 03 B4E1B2_HUMAN  cDNA FLJ53691, highly similar to serotransferrin 142 16 2.78 0.59 6.58 E - 09 B7Z8B6_HUMAN  cDNA FLJ54395, highly similar to inter-alpha-trypsin inhibitor heavy chain H1 68 8 1.2 0.71 6.32 E - 06 BIG3_HUMAN ARFGEF3 Brefeldin A-inhibited guanine nucleotide-exchange protein 3 3 1 0.01 0.71 7.55 E - 03 BLVRB_HUMAN BLVRB Flavin reductase (NADPH) 6 2 0.28 0.81 1.46 E - 02 CATA_HUMAN CAT Catalase 3 3 0.15 0.80 4.91 E - 03 CATF_HUMAN CTSF Cathepsin F 6 4 0.23 0.81 2.58 E - 03 CO2_HUMAN C2 Complement C2 20 9 0.5 0.78 1.25 E - 03 CO5_HUMAN C5 Complement C5 416 52 4.64 0.74 4.50 E - 04 F13B_HUMAN F13B Coagulation factor XIII B chain 4 3 0.15 0.76 1.83 E - 03 GLUC_HUMAN GCG Glucagon 2 1 0.3 0.81 8.52 E - 03 H0YLF3_HUMAN B2M Beta-2-microglobulin (fragment) 4 1 0.75 0.77 5.98 E - 03 HEMO_HUMAN HPX Hemopexin 63 9 1.51 0.65 4.59 E - 08 HGFA_HUMAN HGFAC Hepatocyte growth factor activator 15 4 0.39 0.82 4.99 E - 03 HV306_HUMAN  Ig heavy chain V-III region BUT 36 5 6.45 0.76 2.60 E - 04 HV314_HUMAN  Ig heavy chain V-III region LAY 3 2 0.88 0.81 8.62 E - 03 HV317_HUMAN  Ig heavy chain V-III region ZAP 3 1 0.56 0.78 1.26 E - 03 IC1_HUMAN SERPING1 Plasma protease C1 inhibitor 34 7 0.82 0.62 1.32 E - 09 INHBC_HUMAN INHBC Inhibin beta C chain 16 3 0.36 0.82 1.47 E - 02 ITIH1_HUMAN ITIH1 Inter-alpha-trypsin inhibitor heavy chain H1 252 22 3 0.81 1.20 E - 02 K1C10_HUMAN KRT10 Keratin, type I cytoskeletal 10 32 8 0.87 0.70 1.75 E - 06 K1C14_HUMAN KRT14 Keratin, type I cytoskeletal 14 3 2 0.11 0.68 1.74 E - 06 K1C9_HUMAN KRT9 Keratin, type I cytoskeletal 9 24 8 0.65 0.81 5.29 E - 03 K22E_HUMAN KRT2 Keratin, type II cytoskeletal 2 epidermal 38 14 1.31 0.77 4.04 E - 04 K2C1_HUMAN KRT1 Keratin, type II cytoskeletal 1 104 19 4.31 0.66 1.81 E - 08 K2C6B_HUMAN KRT6B Keratin, type II cytoskeletal 6B 18 4 0.51 0.63 3.76 E - 09 K7ER74_HUMAN APOC4-APOC2 Protein APOC4-APOC2 111 5 7.56 0.68 7.94 E - 05 KV201_HUMAN  Ig kappa chain V-II region Cum 23 2 1.98 0.54 2.24 E - 09 L0R8K6_HUMAN TRIM25 Alternative protein TRIM25 13 1 0.9 0.77 8.84 E - 04 MASP1_HUMAN MASP1 Mannan-binding lectin serine protease 1 36 7 0.63 0.83 1.78 E - 02 PCDGG_HUMAN PCDHGB4 Protocadherin gamma-B4 3 1 0.03 0.80 4.10 E - 02 PLA1A_HUMAN PLA1A Phospholipase A1 member A 2 1 0.07 0.83 5.39 E - 03 PROC_HUMAN PROC Vitamin K-dependent protein C 84 9 1.71 0.78 1.40 E - 03 Q06AH7_HUMAN TF Transferrin 141 16 2.64 0.59 6.40 E - 09 Q1T720_HUMAN HLA-B MHC class I antigen (fragment) 9 6 0.9 0.77 1.30 E - 03 Q6U2L6_HUMAN C4B C4B (fragment) 79 6 27.02 0.69 2.60 E - 05 Q9P1C5_HUMAN  PRO2769 43 7 0.97 0.70 1.53 E - 06 Q9UMV1_HUMAN C4B Complement C4B1a (fragment) 8 1 1.56 0.77 3.10 E - 04 Q9UNU2_HUMAN C4B Complement protein C4B frameshift mutant (fragment) 524 14 26.83 0.64 8.08 E - 07 QPCT_HUMAN QPCT Glutaminyl-peptide cyclotransferase 7 4 0.43 0.83 1.22 E - 02 TIMP3_HUMAN TIMP3 Metalloproteinase inhibitor 3 2 1 0.11 0.75 1.01 E - 03 F o l d   a change provided by MASCOT. p   b values calculated by edgeR to show the significance of different expression.Compared to HCTL group up- and downregulated proteins exclusively in LSSD-CHB and DHSM-CHB patients showed their potential ability of being biomarkers for these two subtypes of HBV induced CHB. Some of them have been reported in other studies; however, more experiments need to be performed to investigate their functions and validate their specificity and accuracy in clinical trials.


### 3.6. Validation of the Quantitative Proteomic Analysis

To validate the results obtained by proteomics analysis, eight randomly selected proteins and internal control albumin with altered expression profile were monitored by western blotting in an independent group of samples. Figures4(a) and 4(b) showed the western blots for eight proteins and internal control albumin. PSMA6 (20S proteasome alpha6), PSMA7 (20S proteasome alpha7/alpha8) were upregulated and PF4 V (platelet factor 4 variant) was downregulated in LSSD-CHB group compared to HCTL (Figure 4(c)). Except SERPING1 (plasma protease C1 inhibitor), AHSG (fetuin-A), ACTB (actin), CTSC (cathepsin C), and PLTP (phospholipid transfer protein) were upregulated in the serum of DHSM-CHB patients (Figure 4(d)). Although the difference between patients and healthy participants was not significant by western blotting analysis, their regulations in patients and healthy group were consistent with iTRAQ. The original images of western blots (see Figure S1) might contain some differences due to brightness and contrast settings.Figure 4
Western blot analysis. (a) Western blot analysis for PSMA7, PSMA6, and PF4 V in LSSD-CHB patients. (b) Western blot analysis for AHSG, ACTB, CTSC, PLTP, and SERPING1 in DHSM-CHB patients. Box plots of the relative intensity of candidate proteins in LSSD-CHB (c) and DHSM-CHB (d) groups.
(a) (b) (c) (d)


## 3.1. Diagnosis of the Patients

To study serum protein biomarkers in LSSD and DHSM CHB patients, we obtained a total of 80 participants, including 30 LSSD-CHB, 30 DHSM-CHB patients, and 20 healthy volunteers. As shown in Table1 and Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/3290260, mean ages of LSSD-CHB, DHSM-CHB, and HCTL were 30, 36.8, and 35.5 years, respectively. Except missing information of three, all patients were positive to HBsAg and anti-HBc. There were two LSSD-CHB patients positive to anti-HBs, and 9 LSSD-CHB and 12 DHSM-CHB patients positive to anti-HBe. HBV-DNA levels in the blood samples of LSSD-CHB and DHSM-CHB patients were ranged from 5.12E+03~1.12E+08 IU/mL and 6.34E+04~9.40E+08 IU/mL, respectively. It is interesting that hepatitis B viral load (HBV-DNA copies) was significantly different (p=0.0096) in LSSD-CHB and DHSM-CHB patients. Next, we examined ALT, AST, STB, CB, and UCB levels in the blood samples of CHB patients. Mean values of these diagnosis biomarkers in the blood samples of LSSD-CHB and DHSM-CHB patients were similar. In addition, the levels of ALT and AST remained at a high level, compared to healthy individuals [26, 27], which confirmed their CHB infection.Table 1
Clinical diagnosis of patients who participated in this study.
Diagnosis Unit LSSD-CHB (n=30) DHSM-CHB (n=30) HCTL (n=20) p value Sex Male  24 15 10 Female  6 15 10  Age Years 17~56 18~60 24~56 Mean age 30.8 36.83 36.15 Standard deviation (SD) 10.526 11.885 11.554  Hepatitis B surface antigen (HBsAg) Positive  28 29 Negative  0 0  Hepatitis surface antibody (anti-HBs) Positive  2 0 Negative  25 29  Hepatitis Be antigen (HBeAg) Positive  21 19 Negative  7 9  Hepatitis Be antibody (anti-HBe) Positive  9 12 Negative  19 16  Hepatitis B core antibody (anti-HBc) Positive  28 29 Negative  0 0  Alanine transaminase (ALT) IU/L 13.8~627 34~673  1.0 Mean ALT level  190.153 187.393 SD  161.231 177.618  Aspartate aminotransferase (AST) IU/L 26.5~345 28~556  0.9999 Mean AST  96.74 142.427 SD  71.699 153.539  Serum total bilirubin (STB) umol/L 10.1~48.63 6.5~109.5  1.0 Mean STB  19.208 20.643 SD  8.926 18.127  Conjugated bilirubin (CB) umol/L 2.4~16.4 2.3~99.2  1.0 Mean CB  6.923 9.408 SD  3.542 17.208  Unconjugated bilirubin (UCB) umol/L 6~34.13 3.3~29.3  1.0 Mean UCB  12.284 11.182 SD  5.951 4.921  HBV-DNA IU/mL 5.12 E + 03~1.12E+08 6.34 E + 04~9.40E+08  0.0096 Mean HBV-DNA  2.521 E + 07 5.655 E + 07 SD  3.221 E + 07 1.717 E + 08


## 3.2. Protein Identification and Quantification by iTRAQ

Next, we quantified the serum proteins in these LSSD-CHB and DHSM-CHB patients using iTRAQ. Initially, a total of 371,034 spectra were generated by liquid chromatography coupled to mass spectrometry (LC-MS/MS) analysis. Of them, 98,243 spectra (5,591 unique peptides) were aligned to 842 proteins from 666 families. The mass distribution of identified proteins (Figure1(a)) suggested by Mascot revealed 170 (98.69%) were above 10 kDa, of which 170 (20.19%) and 141 (16.75%) were 10 to 20 kDa and above 100 kDa, respectively. We also counted the proteins aligned with significant peptides, shown in Figure 1(b), and 547 (64.96%) proteins were aligned by two and more peptides. In addition, the distribution of protein sequence coverage is shown in Figure 1(c). Protein sequence coverage with 40~100%, 30~40%, 20~30%, 10~20%, and under 10% variation accounted for 8.79%, 14.25%, 17.70%, 23.28%, and 35.99%, respectively. In Figure 1(d), we showed correlation between two samples and found LSSD-CHB samples were closer to DHSM-CHB samples than HCTL.Figure 1
Identification and analysis of serum proteome of CHB infected patients. (a) Distribution of protein mass of identified proteins. (b) Number of peptides that match to proteins as indicated by MASCOT 2.4.0. (c) Coverage of identified proteins in CHB patient serum. (d) Correlation between samples calculated by corrplot.
(a) (b) (c) (d)


## 3.3. Identification of Differentially Expressed Proteins

Differentially expressed proteins were defined as those showed greater than 1.2-fold change in relative abundance and ap value < 0.05. Compared to HCTL we identified a total of 392 proteins differentially expressed (Table S2), of which 273 were identified in LSSD-CHB group and 345 in DHSM-CHB group. As shown in the volcano plots, we identified 172 upregulated and 101 downregulated proteins in LSSD-CHB group (Figure 2(a)) and 199 upregulated and 146 downregulated proteins in DHSM-CHB group (Figure 2(b)), compared to HCTL group. Venn diagram (Figure 2(c)) revealed LSSD-CHB and DHSM-CHB shared 142 upregulated and 84 downregulated proteins; 30 and 57 proteins were exclusively upregulated in LSSD-CHB and DHSM-CHB, respectively; 17 and 62 proteins were exclusively downregulated in LSSD-CHB and DHSM-CHB, respectively; and no protein was identified with upregulation in one CHB subtype but with downregulation in another.Figure 2
Identification of differentially expressed proteins in LSSD-CHB and DHSM-CHB patients compared with HCTL. (a) Volcano plot of differentially expressed proteins between LSSD-CHB and HCTL groups. (b) Volcano plot of differentially expressed proteins between DHSM-CHB and HCTL groups. (c) Venn diagram of up- and downregulated proteins in LSSD-CHB and DHSM-CHB groups.
(a) (b) (c)


## 3.4. Potential Biomarkers for CHB

The identification of proteins differentially expressed in LSSD-CHB and DHSM-CHB groups relative to the HCTL group was of interest as these could provide leads for potentially useful diagnostic and prognostic biomarkers. First, we examined those 142 commonly upregulated and 84 commonly downregulated proteins. As shown in Table2, the largest upregulated protein family was immunoglobulin related protein, showing 20 upregulated and 3 downregulated proteins identified. In clinical immunology, levels of immunoglobulins especially IgG can be used to characterize viral hepatitis in patients [28, 29]. Four IgG subclasses (IgG1 to IgG4) differ in their heavy chain constant regions and have different effects on virus-cell fusion inhibition, virus neutralization, and overall course of infection, as have been reported for various viruses including HIV [30] and HBV [31]. Highly expressed proteins encoding heavy chains for immunoglobulins including IGHG1, IGHG3, IGHG4, and IGH@ have been reported with upregulation in HBV [32, 33] and HCC patients [34]. Other upregulated protein families such as heat shock protein, histone, ras-related protein, and von Willebrand factor identified in current study have also been reported in patients infected by HBV or hepatitis C virus (HCV) [35–38]. The largest downregulated protein family was complement, 15 complement proteins downregulated in LSSD-CHB with 0.82- to 0.37-fold change and in DHSM-CHB with 0.77- to 0.31-fold change. Other protein families like insulin-like growth factor binding protein and serum amyloid protein were also decreased in CHB patients in comparison to HCTL group. In addition, several known upregulated proteins from other families in patients infected by HBV or HCV (Table 2), such as apolipoproteins (APOA2, APOB, and APOB-variant) [39, 40], A2M (alpha-2-macroglobulin) [41], alpha-actinin-3 (ACTN3) [42, 43], vimentin (VIM) [38], and putative uncharacterized proteins (DKFZp686N02209 and DKFZp686I04196) [34, 44, 45], were identified in LSSD-CHB and DHSM-CHB groups. The different expression of proteins in the serum of CHB patients indicates they may have functions in response of HBV and CHB processing and can be used as biomarkers in clinical diagnosis.Table 2
Commonly up- and downregulated proteins in LSSD-CHB and SSDHS-CHB groups (part).
Family UniProt_ID Gene_name Description Number of significant matches Number of significant sequences emPAI LSSD-CHB versus HCTL SSDHS-CHB versus HCTL F C a p   v a l u e b F C a p   v a l u e b Immunoglobulin related proteins A0A087WV47_HUMAN IGHG1 Ig gamma-1 chain C region 1541 14 5.37 1.84 6.30 E - 05 1.47 2.71 E - 04 A0A087WYC5_HUMAN IGHG1 Ig gamma-1 chain C region 1501 14 4.82 1.87 3.51 E - 05 1.48 1.69 E - 04 A0A087X1C7_HUMAN IGHG1 Ig gamma-1 chain C region 1511 13 4.79 1.84 6.12 E - 05 1.47 2.60 E - 04 A0A087WXL8_HUMAN IGHG3 Ig gamma-3 chain C region 739 11 2.07 1.84 5.45 E - 05 1.47 1.12 E - 04 A0A0G2JPD4_HUMAN IGHG4 Ig gamma-4 chain C region (fragment) 31 6 1.56 1.69 6.47 E - 04 1.24 4.12 E - 02 KV112_HUMAN  Ig kappa chain V-I region Kue 2 2 0.53 1.82 1.39 E - 03 1.51 9.53 E - 04 KV311_HUMAN  Ig kappa chain V-III region IARC/BL41 2 1 0.49 1.61 5.86 E - 03 1.28 1.64 E - 02 LV302_HUMAN  Ig lambda chain V-III region LOI 25 3 4.01 1.73 1.75 E - 04 1.35 8.79 E - 04 LV301_HUMAN  Ig lambda chain V-III region SH 6 1 0.61 1.99 1.59 E - 07 1.56 5.32 E - 07 S6B2B6_HUMAN  IgG H chain 384 3 0.6 2.05 1.06 E - 04 1.57 9.46 E - 04 S6BAM6_HUMAN  IgG H chain 389 4 0.9 2.05 4.60 E - 05 1.56 5.55 E - 04 S6BGE0_HUMAN  IgG H chain 394 4 1.13 2.04 4.84 E - 05 1.56 6.39 E - 04 S6C4S4_HUMAN  IgG H chain 384 3 0.73 2.05 9.63 E - 05 1.57 5.91 E - 04 S6C4R7_HUMAN  IgG L chain 257 6 6.68 1.53 2.61 E - 02 1.27 4.54 E - 02 Q6GMX6_HUMAN IGH@ IGH@ protein 1524 14 6.04 1.83 6.27 E - 05 1.46 3.15 E - 04 A0A0F7TC28_HUMAN IGHV4-4 IGHV4-4 protein (fragment) 3 2 1.27 1.49 9.67 E - 03 1.24 2.27 E - 02 Q5FWF9_HUMAN IGL@ IGL@ protein 143 6 6.03 1.60 1.35 E - 02 1.32 2.83 E - 02 Q6PIK1_HUMAN IGL@ IGL@ protein 197 5 4.45 1.59 1.37 E - 02 1.29 4.62 E - 02 Q9UL84_HUMAN  Myosin-reactive immunoglobulin heavy chain variable region (fragment) 6 3 1.69 1.52 1.58 E - 02 1.46 2.04 E - 04 Q9UL79_HUMAN  Myosin-reactive immunoglobulin light chain variable region (fragment) 2 1 0.57 0.65 6.56 E - 04 0.50 1.93 E - 12 A0A0C4DH33_HUMAN IGHV1-24 Protein IGHV1-24 (fragment) 5 2 0.84 0.82 5.43 E - 03 0.81 1.38 E - 02 A0A0A0MT74_HUMAN IGKV1-16 Protein IGKV1-16 (fragment) 5 1 0.9 1.58 3.55 E - 03 1.26 3.22 E - 02 A0A075B6I9_HUMAN IGLV7-46 Protein IGLV7-46 (fragment) 31 3 1.41 1.54 9.85 E - 03 1.36 2.46 E - 03  Complement B7Z1F8_HUMAN  cDNA FLJ53025, highly similar to complement C4-B 2230 12 56.68 0.78 1.27 E - 03 0.77 2.33 E - 03 B4E3S6_HUMAN  cDNA FLJ58413, highly similar to complement component C7 63 10 1.74 0.55 8.69 E - 10 0.50 8.03 E - 15 A8K2T4_HUMAN  cDNA FLJ78207, highly similar to human complement protein component C7 mRNA 86 15 1.31 0.55 1.62 E - 09 0.50 2.39 E - 13 B2RA39_HUMAN  cDNA, FLJ94686, highly similar toHomo sapiens complement factor H-related 5 (CFHL5), mRNA 35 5 0.51 0.54 7.03 E - 12 0.57 1.44 E - 11 CO4A_HUMAN C4A Complement C4-A 5889 65 21.28 0.79 2.87 E - 03 0.75 7.70 E - 04 CO4B_HUMAN C4B Complement C4-B 5983 66 23.03 0.79 2.95 E - 03 0.74 5.91 E - 04 A0A024R035_HUMAN C9 Complement component 9, isoform CRA_a 225 17 5.12 0.67 1.36 E - 05 0.54 6.61 E - 13 CO6_HUMAN C6 Complement component C6 92 12 0.83 0.50 3.34 E - 12 0.48 4.06 E - 15 CO8A_HUMAN C8A Complement component C8 alpha chain 66 8 1.51 0.66 1.94 E - 06 0.53 8.36 E - 15 F5GY80_HUMAN C8B Complement component C8 beta chain 133 16 3.28 0.65 6.59 E - 07 0.61 1.07 E - 09 CO8G_HUMAN C8G Complement component C8 gamma chain 41 8 6.86 0.61 8.80 E - 08 0.50 3.38 E - 14 B1AKG0_HUMAN CFHR1 Complement factor H-related protein 1 216 6 4.26 0.66 1.30 E - 06 0.75 4.80 E - 04 FHR3_HUMAN CFHR3 Complement factor H-related protein 3 45 6 1.19 0.82 7.05 E - 03 0.77 2.36 E - 03 FHR4_HUMAN CFHR4 Complement factor H-related protein 4 31 3 0.41 0.37 3.90 E - 23 0.31 1.62 E - 44 A0A0S2Z4I5_HUMAN CFP Complement factor properdin isoform 1 (fragment) 13 2 0.17 0.58 2.21 E - 11 0.72 2.90 E - 05  Apolipoprotein APOA2_HUMAN APOA2 Apolipoprotein A-II 421 2 1.84 1.48 4.64 E - 02 1.26 3.72 E - 02 A0A0B4RUS7_HUMAN APOA5 Apolipoprotein A-V, isoform CRA_a 15 7 1 0.73 2.03 E - 05 0.62 2.95 E - 08 E1B4S9_HUMAN APOB Apolipoprotein B (fragment) 129 7 10.94 1.97 1.23 E - 05 1.53 9.70 E - 05 C0JYY2_HUMAN APOB Apolipoprotein B (including Ag(X) antigen) 5227 215 21.89 1.84 2.08 E - 04 1.44 1.07 E - 03 Q59HB3_HUMAN  Apolipoprotein B variant (fragment) 1661 69 16.33 1.85 2.79 E - 04 1.41 1.40 E - 03 B0YIW2_HUMAN APOC3 Apolipoprotein C-III 577 5 8.48 0.66 1.44 E - 05 0.55 3.02 E - 09 APOC4_HUMAN APOC4 Apolipoprotein C-IV 26 5 3.84 0.80 1.35 E - 03 0.60 8.22 E - 09  Histone H15_HUMAN HIST1H1B Histone H1.5 4 2 0.35 2.02 8.32 E - 06 1.72 1.02 E - 04 A0A024R017_HUMAN HIST1H2AC Histone H2A 21 4 2.27 4.67 1.78 E - 28 3.63 4.54 E - 33 C9J0D1_HUMAN H2AFV Histone H2A 13 3 1.11 4.84 3.09 E - 29 3.73 2.99 E - 37 A0A024QZZ7_HUMAN HIST1H2BD Histone H2B 12 4 1.15 4.58 1.39 E - 29 3.38 6.00 E - 31 B2R4P9_HUMAN H3F3A Histone H3 9 3 0.88 4.98 5.44 E - 32 3.72 9.13 E - 39 Q5TEC6_HUMAN HIST2H3PS2 Histone H3 4 3 0.6 2.59 1.10 E - 08 1.87 3.39 E - 06 B2R4R0_HUMAN HIST1H4J Histone H4 39 5 13.27 4.47 3.83 E - 35 3.41 3.84 E - 45  Heat shock protein A0A024RD80_HUMAN HSP90AB1 Heat shock protein 90 kDa alpha (cytosolic), class B member 1, isoform CRA_a 12 6 0.31 1.46 3.59 E - 02 1.51 2.77 E - 05 HS90A_HUMAN HSP90AA1 Heat shock protein HSP 90-alpha 28 10 0.55 1.58 5.45 E - 03 1.65 3.81 E - 07  Insulin-like growth factor binding protein C1K3N3_HUMAN IGFBP1 Insulin-like growth factor binding protein 1 (fragment) 3 2 0.22 0.61 2.62 E - 07 0.60 5.40 E - 09 A0A024R1U8_HUMAN IGFBP4 Insulin-like growth factor binding protein 4, isoform CRA_a 2 1 0.1 0.66 8.52 E - 06 0.66 1.00 E - 06 A0A024R433_HUMAN IGFBP5 Insulin-like growth factor binding protein 5, isoform CRA_a 3 2 0.18 0.83 3.37 E - 03 0.80 4.98 E - 02  Ras-related protein A0A024R7I3_HUMAN RAB8A RAB8A, member RAS oncogene family, isoform CRA_a 11 4 1.08 1.75 1.87 E - 03 1.73 9.62 E - 06 A0A024RB87_HUMAN RAP1B RAP1B, member of RAS oncogene family, isoform CRA_a 10 4 1.66 1.85 8.55 E - 05 1.96 7.63 E - 11 RAB10_HUMAN RAB10 Ras-related protein Rab-10 9 3 0.71 1.74 2.50 E - 03 1.72 2.60 E - 05 RB11B_HUMAN RAB11B Ras-related protein Rab-11B 5 4 0.73 1.91 3.03 E - 05 1.57 1.03 E - 05 RAB1B_HUMAN RAB1B Ras-related protein Rab-1B 12 6 1.5 1.73 2.07 E - 03 1.73 7.38 E - 06 RB27B_HUMAN RAB27B Ras-related protein Rab-27B 5 4 0.7 1.70 1.33 E - 03 1.59 2.26 E - 05 RAB7A_HUMAN RAB7A Ras-related protein Rab-7a 16 6 1.94 1.60 2.16 E - 02 1.66 1.48 E - 04  Serum amyloid D3DQX7_HUMAN SAA1 Serum amyloid A protein 59 5 9.88 0.53 2.58 E - 13 0.40 3.36 E - 24 SAA1_HUMAN SAA1 Serum amyloid A-1 protein 40 4 4.99 0.48 1.21 E - 15 0.38 4.55 E - 28 SAA2_HUMAN SAA2 Serum amyloid A-2 protein 24 4 4.99 0.48 7.27 E - 16 0.36 3.08 E - 31 SAMP_HUMAN APCS Serum amyloid P-component 228 8 15.23 0.83 8.13 E - 03 0.70 4.45 E - 05  von Willebrand factor L8E853_HUMAN VWF von Willebrand factor 189 38 0.98 1.68 2.69 E - 03 1.41 9.67 E - 04 VWF_HUMAN VWF von Willebrand factor 186 38 0.93 1.67 3.05 E - 03 1.41 7.94 E - 04 F o l d   a change provided by MASCOT. p   b values calculated by edgeR to show the significance of different expression.We next analyzed the potential functions of commonly differentially expressed serum proteins in LSSD-CHB and DHSM-CHB groups using DAVID Bioinformatics Resources 6.7 [24] and STRING v10 [25]. Cellular component annotation (Figure 3(a)) showed 63 and 7 proteins were “extracellular region” (GO: 0005576, GO: 0005615, and GO: 0044421) and “lipids” (GO: 0032994 and GO: 0034358), respectively. However biological process annotation (Figure 3(b)) showed most of the differentially expressed proteins associated with immune response, including “acute inflammatory response” (GO: 0002526), “response to wounding” (GO: 0009611), “inflammatory response” (GO: 0006954), “complement activation” (GO: 0006956), “defense response” (GO: 0006952), “humoral immune response mediated by circulating immunoglobulin” (GO: 0002455), “immune effector process” (GO: 0002252), “B cell mediated immunity” (GO: 0019724), and “activation of immune response” (GO: 0002253). It has been well studied that immunological events are necessary to control hepatitis B virus (HBV) infection [46, 47]. In addition, KEGG pathway analysis also showed differentially expression proteins function mainly in the pathways of “complement and coagulation cascades” (hsa04610), “systemic lupus erythematosus” (hsa05322), “focal adhesion” (hsa04510), and “viral carcinogenesis” (hsa05203). Overall, differentially expressed proteins in both LSSD-CHB and DHSM-CHB groups have potential ability to be used as biomarkers.Figure 3
Gene Ontology annotation for shared differentially expressed proteins in LSSD- and DHSM-CHB groups: (a) cellular component, (b) biological process.
(a) (b)


## 3.5. Dysregulated Proteins Detected Exclusively in LSSD-CHB and DHSM-CHB

Next, we examined differentially expressed proteins exclusively in LSSD-CHB and DHSM-CHB groups. A total of 30 upregulated and 17 downregulated proteins were specifically identified in LSSD-CHB patient serum samples (Table3). Among them 11 upregulated immunoglobulin related proteins, gelsolin (GSN), serum paraoxonase/lactonase 3 (PON3), likely SNC73 protein, transient receptor potential cation channel subfamily M member 8 (TRPM8), and several uncharacterized proteins (DKFZp686M08189, DKFZp686C02220, and DKFZp686K04218) attracted our attention due to their high abundance. Serum PON3 concentrations have been reported to increase in patients with CHB or cirrhosis and showed significant direct correlations with the degree of periportal abnormalities including fibrosis and with serum FAS (a marker of antiapoptosis) concentrations [48]; however, serum gelsolin level has been reported to reduce significantly in patients with acute liver failure (47%), myocardial infarction (69%), sepsis (51%), and myonecrosis (66%) [49]. Among the specifically downregulated serum proteins in LSSD-CHB patients fibulin-1 (FBLN1) is a tumor suppressor in hepatocellular carcinoma [50]. Proteins specifically differentially expressed in LSSD-CHB patients were predicted to function mainly in biological processes of “protein activation cascade” (GO: 0072376), “regulation of response to wounding” (GO: 1903034), “blood coagulation, fibrin clot formation” (GO: 0072378), “negative regulation of response to stimulus” (GO: 0048585), and “acute-phase response” (GO: 0006953).Table 3
Up- and downregulated serum proteins specifically in LSSD-CHB.
UniProt_ID Gene_name Description Number of significant matches Number of significant sequences emPAI F C a p   v a l u e b Upregulated A0A024R6I9_HUMAN SERPINA4 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 4, isoform CRA_a 79 12 3.79 1.78 5.24 E - 05 A0A075B6K3_HUMAN IGLV2-11 Protein IGLV2-11 (fragment) 8 1 0.9 1.43 4.94 E - 02 A0A075B6K6_HUMAN IGLV4-3 Protein IGLV4-3 4 1 0.51 1.54 1.23 E - 02 A0A075B6N7_HUMAN IGHA2 Ig alpha-2 chain C region (fragment) 80 6 1.94 1.44 4.71 E - 02 A0A0A0MS51_HUMAN GSN Gelsolin 146 16 2.42 1.51 2.86 E - 02 A0A0B4J1V4_HUMAN IGHV1-46 Protein IGHV1-46 (fragment) 2 1 0.23 1.44 4.22 E - 02 A0N7J6_HUMAN  REV25-2 (fragment) 2 2 0.47 1.92 3.69 E - 03 A0N8J1_HUMAN  V(k)3 sequence of NG9 gene from fetal liver DNA (fragment) 6 1 1.1 1.49 1.49 E - 02 CPN2_HUMAN CPN2 Carboxypeptidase N subunit 2 94 11 2.09 1.48 1.88 E - 02 D3DNU8_HUMAN KNG1 Kininogen 1, isoform CRA_a 91 14 3.3 1.50 1.14 E - 02 FETUA_HUMAN AHSG Alpha-2-HS-glycoprotein 57 3 0.64 1.70 1.36 E - 03 FETUB_HUMAN FETUB Fetuin-B 9 3 0.29 1.65 6.32 E - 04 HV208_HUMAN  Ig heavy chain V-II region SESS 2 1 0.21 2.27 1.25 E - 08 ITB1_HUMAN ITGB1 Integrin beta-1 3 2 0.06 1.46 3.43 E - 02 KV119_HUMAN  Ig kappa chain V-I region Wes 21 2 2.16 1.52 6.37 E - 03 KV308_HUMAN  Ig kappa chain V-III region CLL 4 1 0.49 1.65 8.16 E - 03 LV204_HUMAN  Ig lambda chain V-II region TRO 7 2 0.96 1.59 1.25 E - 02 PCYOX_HUMAN PCYOX1 Prenylcysteine oxidase 1 48 9 1.89 1.43 3.70 E - 02 PEDF_HUMAN SERPINF1 Pigment epithelium-derived factor 23 9 1.54 1.48 1.56 E - 02 PON3_HUMAN PON3 Serum paraoxonase/lactonase 3 179 12 3.89 1.51 4.11 E - 02 Q6MZX9_HUMAN DKFZp686M08189 Putative uncharacterized protein DKFZp686M08189 124 7 1.41 1.43 3.95 E - 02 Q6N091_HUMAN DKFZp686C02220 Putative uncharacterized protein DKFZp686C02220 (fragment) 71 6 1.2 1.46 2.31 E - 02 Q6ZVX0_HUMAN  cDNA FLJ41981 fis, clone SMINT2011888, highly similar to protein Tro alpha1 H,myeloma 125 7 1.38 1.43 4.13 E - 02 Q7Z379_HUMAN DKFZp686K04218 Putative uncharacterized protein DKFZp686K04218 (fragment) 124 7 1.44 1.43 3.96 E - 02 Q8TE63_HUMAN  Immunoglobulin light chain variable region (fragment) 4 2 0.97 1.45 4.51 E - 02 Q96K68_HUMAN  cDNA FLJ14473 fis, clone MAMMA1001080, highly similar toHomo sapiens SNC73 protein (SNC73) mRNA 205 9 1.77 1.45 2.93 E - 02 Q9NPP6_HUMAN  Immunoglobulin heavy chain variant (fragment) 125 7 1.75 1.43 4.92 E - 02 Q9UL83_HUMAN  Myosin-reactive immunoglobulin light chain variable region (fragment) 14 2 2.98 1.54 5.43 E - 03 Q9UL89_HUMAN  Myosin-reactive immunoglobulin heavy chain variable region (fragment) 18 5 8.63 1.49 2.16 E - 02 TRPM8_HUMAN TRPM8 Transient receptor potential cation channel subfamily M member 8 3 2 0.04 1.60 6.39 E - 03  Downregulated A0A024QZK7_HUMAN HK1 Hexokinase 5 1.00 E + 00 0.03 0.82 7.31 E - 03 A0A024R2X3_HUMAN HYAL1 Hyaluronidase 12 5.00 E + 00 0.55 0.73 2.82 E - 05 A0A024R451_HUMAN SERPINE2 Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2, isoform CRA_a 17 8 1.05 0.83 2.36 E - 03 A0A024RAG6_HUMAN C1QA Complement component 1, q subcomponent, A chain, isoform CRA_a 12 4 1.08 0.75 4.11 E - 04 A0A0S2Z3Y1_HUMAN LGALS3BP Lectin galactoside-binding soluble 3 binding protein isoform 1 (fragment) 247 14 4.17 0.71 2.49 E - 06 A0A0S2Z4D4_HUMAN PLP1 Proteolipid protein 1 isoform 1 (fragment) 2 1 0.11 0.40 1.65 E - 04 ADT4_HUMAN SLC25A31 ADP/ATP translocase 4 5 1 0.16 0.65 1.28 E - 02 B1AHL2_HUMAN FBLN1 Fibulin-1 74 8 0.92 0.78 4.33 E - 04 CALU_HUMAN CALU Calumenin 12 7 0.86 0.76 2.45 E - 04 CFAH_HUMAN CFH Complement factor H 689 31 4.02 0.83 8.87 E - 03 LBP_HUMAN LBP Lipopolysaccharide-binding protein 148 11 3.18 0.79 3.74 E - 04 LRP1_HUMAN LRP1 Prolow-density lipoprotein receptor-related protein 1 37 20 0.16 0.83 7.44 E - 03 LTBP1_HUMAN LTBP1 Latent-transforming growth factor beta-binding protein 1 19 8 0.16 0.77 8.36 E - 04 NUCB1_HUMAN NUCB1 Nucleobindin-1 17 7 0.66 0.78 5.80 E - 04 Q8NBH6_HUMAN  Fibulin-1 125 11 2.25 0.75 1.26 E - 04 Q9HCC1_HUMAN  Single chain Fv (fragment) 10 2 0.96 0.79 4.29 E - 04 THRB_HUMAN F2 Prothrombin 1145 18 6.65 0.78 6.78 E - 04 F o l d   a change provided by MASCOT. p   b values calculated by edgeR to show the significance of different expression.We also identified 57 upregulated and 62 downregulated proteins exclusively in DHSM-CHB patients (Table4). Two IGL@ proteins (Q6GMX4_HUMAN and Q6PIQ7_HUMAN) were specifically upregulated in DHSM-CHB patients with 1.27-fold change. Transthyretin (TTR), upregulated 1.34-fold in DHSM-CHB, can be induced by hepatitis C virus and activate TGF-β signaling pathway with furin [51]. Interestingly, we found three members of tubulin (TUBA4A, TUBB1, and TUBB8) were upregulated only in DHSM-CHB patients compared with HCTL. Although there are few reports about tubulin and HBV, it is well known that 42 kDa tubulin alpha-6 chain fragment in well-differentiated hepatocellular carcinoma tissues is from patients infected with HCV [52]. In addition, we found actinin, alpha 1 (ACTN1), which can directly interact with HCV [53], GAPDH, which can bind to the HBV posttranscriptional regulatory element [54], and polymeric immunoglobulin receptor (PIGR), the main transporter of IgA [55], were upregulated in DHSM-CHB but not in LSSD-CHB. Among DHSM-CHB specifically downregulated proteins we identified three members of keratin type I (KRT9, KRT10, and KRT14) and another three members of keratin type II (KRT1, KRT2, and KRT6B). Although there is no evidence showing relation between these six keratin proteins with CHB or other liver diseases, variant keratins are associated with progression of fibrosis during chronic hepatitis C infection [56]. Differentially expressed proteins exclusively detected in DHSM-CHB patients were predicted to be involved in the biological processes of “immune system process” (GO: 0002376), “response to stress” (GO: 0006950), “defense response” (GO: 0006952), “immune response” (GO: 0006955), and “single-organism metabolic process” (GO: 0044710).Table 4
Up- and downregulated serum proteins exclusively in DHSM-CHB.
UniProt_ID Gene_name Description Number of significant matches Number of significant sequences emPAI F C a p   v a l u e b Upregulated A0A024R145_HUMAN ALDOB Fructose-bisphosphate aldolase 5 3 0.22 1.29 3.81 E - 02 A0A024R1N1_HUMAN MYH9 Myosin, heavy polypeptide 9, nonmuscle, isoform CRA_a 17 9 0.13 1.43 1.34 E - 03 A0A024R5H8_HUMAN RAB6A RAB6A, member RAS oncogene family, isoform CRA_b 8 3 0.57 1.43 4.84 E - 03 A0A024R694_HUMAN ACTN1 Actinin, alpha 1, isoform CRA_a 20 10 0.42 1.33 1.97 E - 02 A0A024R6G3_HUMAN FBLN5 Fibulin 5, isoform CRA_b 6 3 0.27 1.33 6.31 E - 03 A0A024R9T1_HUMAN hCG_39634 HCG39634, isoform CRA_a 2 2 0.26 1.49 4.90 E - 03 A0A024RDB8_HUMAN HPSE Heparanase, isoform CRA_a 23 6 0.55 1.36 1.03 E - 03 A0A024RDL8_HUMAN ASL Argininosuccinate lyase isoform 1 10 5 0.39 1.65 2.02 E - 07 A0A087WT59_HUMAN TTR Transthyretin 175 7 5.66 1.34 3.42 E - 02 A0A087WUL0_HUMAN TKFC Triokinase/FMN cyclase 3 3 0.15 1.28 9.35 E - 03 A0A0A0MSD0_HUMAN SVEP1 Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 4 3 0.02 1.36 7.51 E - 03 A0A0A0MT32_HUMAN LIPA Lysosomal acid lipase/cholesteryl ester hydrolase 2 1 0.09 1.38 4.78 E - 03 A0A0C4DFP6_HUMAN CRTAC1 Cartilage acidic protein 1 6 5 0.24 1.23 3.57 E - 02 A0A0S2Z3F6_HUMAN CETP Cholesteryl ester transfer protein plasma isoform 1 (fragment) 95 14 3.56 1.36 1.42 E - 03 A0A125QYY9_HUMAN  IBM-B2 heavy chain variable region (fragment) 12 2 0.83 1.42 2.25 E - 03 A0N7I9_HUMAN F5-20 F5-20 (fragment) 4 1 0.47 1.68 2.31 E - 03 A2NYU9_HUMAN  Heavy chain Fab (fragment) 2 1 0.48 1.29 3.92 E - 02 A8K486_HUMAN  Peptidyl-prolyl cis-trans isomerase 2 2 0.32 1.40 4.10 E - 02 ADIPO_HUMAN ADIPOQ Adiponectin 27 5 2.13 1.32 1.12 E - 02 APOF_HUMAN APOF Apolipoprotein F 7 3 0.5 1.29 1.84 E - 02 ASSY_HUMAN ASS1 Argininosuccinate synthase 4 3 0.18 1.49 1.09 E - 04 ATPB_HUMAN ATP5B ATP synthase subunit beta, mitochondrial 11 2 0.22 1.34 3.74 E - 02 B3KTV0_HUMAN  cDNA FLJ38781 fis, clone LIVER2000216, highly similar to HEAT SHOCK COGNATE 71 kDa PROTEIN 5 5 0.21 1.29 3.41 E - 02 B4DQK4_HUMAN  cDNA FLJ53743, highly similar to tubulin alpha-3 chain 21 6 0.81 1.61 1.06 E - 03 B4DVA7_HUMAN  Beta-hexosaminidase 3 2 0.15 1.26 1.84 E - 02 B7Z478_HUMAN PSMB2 Proteasome (prosome, macropain) subunit, beta type, 2, isoform CRA_b 2 1 0.3 1.65 1.96 E - 03 BPIB1_HUMAN BPIFB1 BPI fold-containing family B member 1 11 6 0.51 1.28 1.34 E - 02 BTD_HUMAN BTD Biotinidase 47 6 1.12 1.54 1.69 E - 06 CAMP_HUMAN CAMP Cathelicidin antimicrobial peptide 2 1 0.29 1.43 2.64 E - 03 CAND1_HUMAN CAND1 Cullin-associated NEDD8-dissociated protein 1 2 1 0.02 1.36 2.60 E - 03 CATD_HUMAN CTSD Cathepsin D 2 1 0.13 1.28 3.06 E - 02 DHSO_HUMAN SORD Sorbitol dehydrogenase 4 3 0.32 1.67 2.46 E - 07 FIBA_HUMAN FGA Fibrinogen alpha chain 102 22 2.36 1.36 3.18 E - 03 G3P_HUMAN GAPDH Glyceraldehyde-3-phosphate dehydrogenase 19 7 1.37 1.37 5.07 E - 03 G3V5Z7_HUMAN PSMA6 Proteasome subunit alpha type 7 4 0.58 1.44 9.01 E - 04 HV310_HUMAN  Ig heavy chain V-III region HIL 3 1 0.5 1.48 1.12 E - 03 HV320_HUMAN  Ig heavy chain V-III region GAL 36 3 1.91 1.32 2.19 E - 02 KV404_HUMAN  Ig kappa chain V-IV region B17 34 2 1.45 1.45 4.98 E - 04 LDHB_HUMAN LDHB L-lactate dehydrogenase B chain 4 2 0.33 1.65 2.13 E - 06 M0QZB5_HUMAN PPFIA4 Liprin-alpha-4 (fragment) 2 1 0.03 1.33 2.70 E - 02 MARCO_HUMAN MARCO Macrophage receptor MARCO 2 1 0.11 1.31 2.89 E - 02 MDR3_HUMAN ABCB4 Phosphatidylcholine translocator ABCB4 2 1 0.04 1.32 1.23 E - 02 OTUB1_HUMAN OTUB1 Ubiquitin thioesterase OTUB1 2 1 0.09 1.27 2.92 E - 02 PIGR_HUMAN PIGR Polymeric immunoglobulin receptor 7 4 0.18 1.34 4.27 E - 03 PSA3_HUMAN PSMA3 Proteasome subunit alpha type-3 8 4 0.58 1.31 4.84 E - 02 PSB1_HUMAN PSMB1 Proteasome subunit beta type-1 4 3 0.5 1.47 1.25 E - 03 Q6GMX4_HUMAN IGL@ IGL@ protein 442 6 7.15 1.27 3.98 E - 02 Q6PIQ7_HUMAN IGL@ IGL@ protein 441 6 6.39 1.27 4.86 E - 02 Q6ZNX5_HUMAN  CDNA FLJ26936 fis, clone RCT06808 5 1 0.39 1.78 2.10 E - 08 Q9UL82_HUMAN  Myosin-reactive immunoglobulin light chain variable region (fragment) 46 3 2.11 1.41 1.22 E - 03 QSOX1_HUMAN QSOX1 Sulfhydryl oxidase 1 97 20 2.33 1.24 4.15 E - 02 TBA4A_HUMAN TUBA4A Tubulin alpha-4A chain 24 8 0.86 1.55 1.38 E - 03 TBB1_HUMAN TUBB1 Tubulin beta-1 chain 6 4 0.33 1.30 2.87 E - 02 TBB8_HUMAN TUBB8 Tubulin beta-8 chain 7 5 0.42 1.32 3.10 E - 02 TRML1_HUMAN TREML1 Trem-like transcript 1 protein 3 1 0.09 1.42 1.05 E - 03 TSP1_HUMAN THBS1 Thrombospondin-1 365 34 4.23 1.37 1.66 E - 03 TYPH_HUMAN TYMP Thymidine phosphorylase 3 3 0.19 1.23 4.67 E - 02  Downregulated A0A024CIM4_HUMAN  Carboxylic ester hydrolase 7 3 0.17 0.81 1.43 E - 02 A0A024R6I6_HUMAN SERPINA10 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 10, isoform CRA_b 65 12 2.73 0.75 1.46 E - 04 A0A024R6K8_HUMAN WARS Tryptophanyl-tRNA synthetase, isoform CRA_a 5 2 0.1 0.82 4.87 E - 02 A0A024R853_HUMAN IQCE IQ motif containing E, isoform CRA_b 2 1 0.04 0.74 1.67 E - 03 A0A024R9J3_HUMAN COLEC10 Collectin subfamily member 10 (C-type lectin), isoform CRA_a 4 2 0.28 0.83 2.40 E - 02 A0A075B6S2_HUMAN IGKV2D-29 Protein IGKV2D-29 (fragment) 26 3 2.47 0.77 2.05 E - 03 A0A087X054_HUMAN HYOU1 Hypoxia upregulated protein 1 4 2 0.08 0.58 2.93 E - 08 A0A0A7C3P2_HUMAN HLA-A MHC class I antigen (fragment) 8 3 0.72 0.77 1.24 E - 03 A0A0J9YX35_HUMAN  Uncharacterized protein (fragment) 2 1 0.51 0.74 3.43 E - 03 A0A0X9TD47_HUMAN  MS-D1 light chain variable region (fragment) 30 3 5.49 0.83 1.31 E - 02 A2AP_HUMAN SERPINF2 Alpha-2-antiplasmin 67 10 1.86 0.74 2.85 E - 04 A2J1N4_HUMAN  Rheumatoid factor RF-IP24 (fragment) 10 2 1.08 0.80 4.59 E - 03 A2J1N5_HUMAN  Rheumatoid factor RF-ET6 (fragment) 15 1 1.65 0.79 6.40 E - 04 A2MYD0_HUMAN V1-17 V1-17 protein (fragment) 8 3 1.87 0.83 1.21 E - 02 A2NB45_HUMAN  Cold agglutinin FS-1 L-chain (fragment) 23 2 1.39 0.69 3.83 E - 04 A3RKG7_HUMAN  Coagulation factor VII (fragment) 17 6 2.02 0.77 7.55 E - 04 ALBU_HUMAN ALB Serum albumin 1841 31 17.82 0.67 1.64 E - 06 AMBP_HUMAN AMBP Protein AMBP 34 3 0.75 0.76 4.96 E - 04 ATS13_HUMAN ADAMTS13 A disintegrin and metalloproteinase with thrombospondin motifs 13 22 8 0.26 0.81 6.52 E - 03 B2RBZ5_HUMAN  cDNA, FLJ95778, highly similar toHomo sapiens serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 10 (SERPINA10), mRNA 58 11 2.2 0.76 1.77 E - 04 B4DL32_HUMAN  cDNA FLJ59922, highly similar to keratin, type II cytoskeletal 5 7 2 0.33 0.83 2.87 E - 02 B4DN21_HUMAN  cDNA FLJ53365, highly similar toHomo sapiens fibronectin 1 (FN1), transcript variant 4, mRNA 384 8 6.85 0.77 1.76 E - 03 B4E1B2_HUMAN  cDNA FLJ53691, highly similar to serotransferrin 142 16 2.78 0.59 6.58 E - 09 B7Z8B6_HUMAN  cDNA FLJ54395, highly similar to inter-alpha-trypsin inhibitor heavy chain H1 68 8 1.2 0.71 6.32 E - 06 BIG3_HUMAN ARFGEF3 Brefeldin A-inhibited guanine nucleotide-exchange protein 3 3 1 0.01 0.71 7.55 E - 03 BLVRB_HUMAN BLVRB Flavin reductase (NADPH) 6 2 0.28 0.81 1.46 E - 02 CATA_HUMAN CAT Catalase 3 3 0.15 0.80 4.91 E - 03 CATF_HUMAN CTSF Cathepsin F 6 4 0.23 0.81 2.58 E - 03 CO2_HUMAN C2 Complement C2 20 9 0.5 0.78 1.25 E - 03 CO5_HUMAN C5 Complement C5 416 52 4.64 0.74 4.50 E - 04 F13B_HUMAN F13B Coagulation factor XIII B chain 4 3 0.15 0.76 1.83 E - 03 GLUC_HUMAN GCG Glucagon 2 1 0.3 0.81 8.52 E - 03 H0YLF3_HUMAN B2M Beta-2-microglobulin (fragment) 4 1 0.75 0.77 5.98 E - 03 HEMO_HUMAN HPX Hemopexin 63 9 1.51 0.65 4.59 E - 08 HGFA_HUMAN HGFAC Hepatocyte growth factor activator 15 4 0.39 0.82 4.99 E - 03 HV306_HUMAN  Ig heavy chain V-III region BUT 36 5 6.45 0.76 2.60 E - 04 HV314_HUMAN  Ig heavy chain V-III region LAY 3 2 0.88 0.81 8.62 E - 03 HV317_HUMAN  Ig heavy chain V-III region ZAP 3 1 0.56 0.78 1.26 E - 03 IC1_HUMAN SERPING1 Plasma protease C1 inhibitor 34 7 0.82 0.62 1.32 E - 09 INHBC_HUMAN INHBC Inhibin beta C chain 16 3 0.36 0.82 1.47 E - 02 ITIH1_HUMAN ITIH1 Inter-alpha-trypsin inhibitor heavy chain H1 252 22 3 0.81 1.20 E - 02 K1C10_HUMAN KRT10 Keratin, type I cytoskeletal 10 32 8 0.87 0.70 1.75 E - 06 K1C14_HUMAN KRT14 Keratin, type I cytoskeletal 14 3 2 0.11 0.68 1.74 E - 06 K1C9_HUMAN KRT9 Keratin, type I cytoskeletal 9 24 8 0.65 0.81 5.29 E - 03 K22E_HUMAN KRT2 Keratin, type II cytoskeletal 2 epidermal 38 14 1.31 0.77 4.04 E - 04 K2C1_HUMAN KRT1 Keratin, type II cytoskeletal 1 104 19 4.31 0.66 1.81 E - 08 K2C6B_HUMAN KRT6B Keratin, type II cytoskeletal 6B 18 4 0.51 0.63 3.76 E - 09 K7ER74_HUMAN APOC4-APOC2 Protein APOC4-APOC2 111 5 7.56 0.68 7.94 E - 05 KV201_HUMAN  Ig kappa chain V-II region Cum 23 2 1.98 0.54 2.24 E - 09 L0R8K6_HUMAN TRIM25 Alternative protein TRIM25 13 1 0.9 0.77 8.84 E - 04 MASP1_HUMAN MASP1 Mannan-binding lectin serine protease 1 36 7 0.63 0.83 1.78 E - 02 PCDGG_HUMAN PCDHGB4 Protocadherin gamma-B4 3 1 0.03 0.80 4.10 E - 02 PLA1A_HUMAN PLA1A Phospholipase A1 member A 2 1 0.07 0.83 5.39 E - 03 PROC_HUMAN PROC Vitamin K-dependent protein C 84 9 1.71 0.78 1.40 E - 03 Q06AH7_HUMAN TF Transferrin 141 16 2.64 0.59 6.40 E - 09 Q1T720_HUMAN HLA-B MHC class I antigen (fragment) 9 6 0.9 0.77 1.30 E - 03 Q6U2L6_HUMAN C4B C4B (fragment) 79 6 27.02 0.69 2.60 E - 05 Q9P1C5_HUMAN  PRO2769 43 7 0.97 0.70 1.53 E - 06 Q9UMV1_HUMAN C4B Complement C4B1a (fragment) 8 1 1.56 0.77 3.10 E - 04 Q9UNU2_HUMAN C4B Complement protein C4B frameshift mutant (fragment) 524 14 26.83 0.64 8.08 E - 07 QPCT_HUMAN QPCT Glutaminyl-peptide cyclotransferase 7 4 0.43 0.83 1.22 E - 02 TIMP3_HUMAN TIMP3 Metalloproteinase inhibitor 3 2 1 0.11 0.75 1.01 E - 03 F o l d   a change provided by MASCOT. p   b values calculated by edgeR to show the significance of different expression.Compared to HCTL group up- and downregulated proteins exclusively in LSSD-CHB and DHSM-CHB patients showed their potential ability of being biomarkers for these two subtypes of HBV induced CHB. Some of them have been reported in other studies; however, more experiments need to be performed to investigate their functions and validate their specificity and accuracy in clinical trials.


## 3.6. Validation of the Quantitative Proteomic Analysis

To validate the results obtained by proteomics analysis, eight randomly selected proteins and internal control albumin with altered expression profile were monitored by western blotting in an independent group of samples. Figures4(a) and 4(b) showed the western blots for eight proteins and internal control albumin. PSMA6 (20S proteasome alpha6), PSMA7 (20S proteasome alpha7/alpha8) were upregulated and PF4 V (platelet factor 4 variant) was downregulated in LSSD-CHB group compared to HCTL (Figure 4(c)). Except SERPING1 (plasma protease C1 inhibitor), AHSG (fetuin-A), ACTB (actin), CTSC (cathepsin C), and PLTP (phospholipid transfer protein) were upregulated in the serum of DHSM-CHB patients (Figure 4(d)). Although the difference between patients and healthy participants was not significant by western blotting analysis, their regulations in patients and healthy group were consistent with iTRAQ. The original images of western blots (see Figure S1) might contain some differences due to brightness and contrast settings.Figure 4
Western blot analysis. (a) Western blot analysis for PSMA7, PSMA6, and PF4 V in LSSD-CHB patients. (b) Western blot analysis for AHSG, ACTB, CTSC, PLTP, and SERPING1 in DHSM-CHB patients. Box plots of the relative intensity of candidate proteins in LSSD-CHB (c) and DHSM-CHB (d) groups.
(a) (b) (c) (d)


## 4. Discussion

Quantitation of serum or plasma proteins using comparative proteomics has recently been suggested as a suitable approach for the detection of liver disease biomarkers [17, 57–59]. The iTRAQ technology has been proposed as a powerful alternative to common tools (e.g., ELISA) and a flurry of applications emerged in the literature.In this study, iTRAQ LC–MS/MS proteomics was used to detect serum protein as biomarkers of LSSD-CHB and DHSM-CHB patients. We compared the proteomics profile of LSSD-CHB and DHSM-CHB patients with healthy individuals and indicated 142 upregulated and 84 downregulated proteins shared by these two CHB subtype diseases. Protein-protein interaction network (Figure5) showed several significant proteins might function in response to HBV, such as actins (ACTA2, ACTB, ACTBL2, ACTN3, and ACTN4), apolipoproteins (APOA2, APOA5, APOB, APOC3, and APOC4), heat shock proteins (HSP90AA1 and HSP90AB1), and proteasome subunit proteins (PSMA1 and PSMA4). It has been reported that HBV core proteins can interact with the C-terminal region of actin-binding protein [60] and HBV X protein (HBx) can block filamentous actin bundles by interaction with eEF1A1 (eukaryotic translation elongation factor 1 alpha 1) [61]. In addition, ACTA2 is a marker of hepatitis stellate cells and correlated significantly with necroinflammatory grades and fibrotic stages in CHB or CHC [13]. Apolipoproteins are supposed to enhance the infectivity of hepatitis virus during the infection [39, 62] and are identified to interact with HBx as well [63]. Among the apolipoproteins APOA2 is a considerable biomarker because its expression is increased on both mRNA and protein levels in CHB patients [14, 64]. HBx protein also interacts with heat shock proteins and enhances HBx-mediated apoptosis [65]. A HBV-specific peptide (TVATAMG) is associated with heat shock protein and has potential for engineering tumor vaccines against hepatocellular carcinoma and chronic HBV infection [66]. Heat shock proteins like HSP27, HSP90, and GRP78 are upregulated in HBV related hepatocellular carcinoma, associated with vascular invasion and intrahepatic metastasis and have potential to be prognosis markers [67, 68]. Commonly downregulated complement proteins are important mediators of inflammation and contribute to the regulation of the immune response. C4, a predisposing factor to autoimmune chronic active hepatitis [69], is expressed lowly in chronic hepatitis C patient compared to that in controls [70]. Low serum levels of complement in viral hepatitis are associated with high titers of hepatitis-associated antigen [71]. It is said that complement proteins are related to hepatitis B vaccine and C4AQ0 (mutant C4) probably contribute to inefficient complement activation and failure of B cells to secret anti-HBs [72]. Our results confirmed the potential of previously reported proteins in diagnosis of patients infected by HBV.Figure 5
Protein-protein interaction network of commonly differentially expressed proteins in LSSD-CHB and DHSM-CHB patients compared with HCTL group.LSSD-CHB and DHSM-CHB are two subtypes of CHB according to traditional Chinese medicine pattern classification. In this study we identified 47 and 119 differentially expressed proteins exclusively in LSSD-CHB and DHSM-CHB, respectively, which could be used as biomarkers for LSSD-CHB and DHSM-CHB patients. We showed top 5 highly expressed proteins with different expression in LSSD-CHB and DHSM-CHB patients compared to HCTL group in Figure6. Using relative expression ratio calculated by MASCOT we found mean expression levels of some proteins were close in LSSD-CHB and DHSM-CHB but with different p values, such as CFH (complement factor H), F2 (prothrombin), and FGA (fibrinogen alpha chain). As we know, prothrombin time is one of the markers of liver test; it is usually lower in HBV infected patients than in healthy people and a good marker for liver fibrosis [73, 74]. CFH functions as a cofactor in the inactivation of C3b by factor I [75], which can interact with IgG and is moderately depressed in the serum of patients with viral hepatitis [71]. FGA has a major function in hemostasis as one of the primary components of blood clots [76]. Fibrinogen-like protein 2 (FGL2) has been identified as a potential biomarker for severity of CHC infection [77]. Other proteins also have been reported to be associated with HBV infection. LGALS3BP (lectin galactoside-binding soluble 3 binding protein isoform 1) were downregulated in LSSD-CHB patients (fc = 0.71, p value = 2.49E-06) and DHSM-CHB patients (fc = 0.89, p value = 0.061). Previous studies about LGALS3BP in CHB and HCC found its different expression on transcriptional level [78], while in current study we identified its protein was differentially expressed in CHB patients and had the potential to be a good marker for LSSD-CHB subtype. PON3 (serum paraoxonase/lactonase 3), which was upregulated exclusively in LSSD-CHB, might play a hepatoprotective role against histological alterations and hepatic cell apoptosis leading to liver disease [48].Figure 6
Top 5 highly expressed proteins differentially expressed in LSSD-CHB and DHSM-CHB compared to HCTL. Fold changes (fc) andp values (p) of them were shown in red and blue for LSSD-CHB and DHSM-CHB, respectively.DHSM-CHB specifically differentially expressed proteins like ITIH1 (inter-alpha-trypsin inhibitor heavy chain H1), THBS1 (thrombospondin-1), C5 (Complement C5), and ALB (albumin) have been also reported in hepatitis viral related diseases. The expression level of ITIH1 in HCTL group was similar to that in LSSD-CHB patients (fc = 0.99,p value = 0.227) but was downregulated significantly in DHSM-CHB patients (fc = 0.81, p value = 0.012). The low expression of ITIH1 indicated it can be used to differ DHSM-CHB from LSSD-CHB. In addition, it has been experimented to be downregulated in HCV infected patients [79] and hepatitis C associated hepatocellular carcinoma patients [80]. It is reported that HCV viral proteins act directly or indirectly on THBS1 in TGF-β pathway [81]. By noninvasive imaging the gene expression of THBS1 was upregulated in liver cancer [82]. Interestingly, ALB has been reported as an important factor to score the risk of HCC in CHB patients [83]. In our study, ALB was downregulated in both CHB subtypes but significantly exclusively in DHSM-CHB. Our results confirmed its different expression in CHB patients and revealed that the criteria of ALB expression in CHB patients required more patients and experiments.Due to the fact that hepatitis B viral load in DHSM-CHB patients was significantly higher than that in LSSD-CHB patients, we assume HBV-DNA might be related to CHB patients with different syndromes and it requires further experiments. To our knowledge, this study appears to be the first iTRAQ based approach aimed at identifying leads for potential useful biomarkers of patients of CHB subtypes. The candidates identified in this study await rigorous clinical validation using large cohorts of patient samples and more experimental function analysis.



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