The collection and use of all human pathological specimens for the research were approved by the Ethical Committee of Northwest University (Xi'an, China), the Ethical Committee of Second Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China), and the Ethical Committee of Tangdu Hospital of Fourth Military Medical University (Xi'an, China). Written informed consent was received from participants for the collection of their whole serum. This study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki.

Serum samples were collected from 54 CHB, 47 CHC patients, and 35 healthy volunteers (HV). The diagnosis was based on clinical examination and biochemical tests. Liver function and hematological tests were performed using standard methods in a clinical setting. Hepatitis B markers were tested using a commercial radioimmunoassay (Kechuang, Shenzhen, China). HBV DNA levels were quantified using a real-time polymerase chain reaction assay (Qiagen, Shenzhen, China). HBV and HCV infected patients were tested for HBsAg and anti-HCV antibody respectively for more than 6 months. Exclusion criteria were coinfection with HCV or HBV and main complications of liver disease such as alcoholic hepatitis, drug-related hepatitis, and obstructive jaundice. Patients with CHB were positive for hepatitis B surface antigen and presented serum alanine aminotransferase (ALT) levels 2–10 times the upper limits of normal (ULN). Liver fibrosis stages were determined by using non-invasive transient elastography and serological marker-based algorithm. Patients were enrolled in the study performed mild fibrosis (F0–F1) and the detailed information of patients is described in Table 1.

Serum samples were immediately isolated from the clotted whole blood by centrifugation at 1,300 × g for 10 min at 4°C, which was immediately used or stored at −80°C. To normalize the differences between subjects and to tolerate individual variation, 5 μL of serum samples from all HV, CHB, and CHC patients were pooled, respectively. Then, 15, 22, and 23 individual serum samples of the HV, CHB and CHC patients were randomly selected for further validation. The protein concentration was determined by the Bradford protein assay. The pooled and individual serum samples were labeled with Cy3 fluorescent dye and purified using a Sephadex G-25 columns (Yu et al., 2012).

A lectin microarray was produced using 37 lectins [purchased from Vector Laboratories, Sigma-Aldrich (St Louis, MO) and Calbiochem (Billerica, MA)] with different binding preferences covering N- and O-linked glycan according to our previous protocol (Qin et al., 2013). Briefly, 37 lectins were dissolved in the manufacturer's recommended buffer containing 1 mmol/L of the appropriate monosaccharide at a concentration of 1 mg/mL and were spotted on the homemade epoxysilane-coated slides with Stealth micro-spotting pins (SMP-10B; TeleChem, Sunnyvale, CA) by a Capital smart microarrayer (CapitalBio, Beijing, China). Each lectin was spotted in triplicate per block with quadruplicate blocks on one slide. After immobilization, the slides were blocked with the blocking buffer containing 2% (w/v) BSA in 1× phosphate buffer saline (PBS) (0.01 mol/L phosphate buffer containing 0.15 mol/L NaCl, pH 7.4) for 1 h, then rinsed twice with 1× PBST (0.2% Tween 20 in 0.01 mol/L phosphate buffer containing 0.15 mol/L NaCl, pH 7.4) and 1× PBS. Then the appropriate amount of Cy3-labeled protein diluted in 0.5 mL of incubation buffer containing 2%(w/v) bovine serum albumin (BSA), 500 mM glycine, and 0.1% Tween-20 in 1× PBS was applied to the blocked lectin microarrays, and an incubation was performed in the chamber at 37°C for 3 h in a rotisserie oven set at 4 rpm. The slide was washed with 1× PBST twice for each for 5 min and washed once with 1× PBS for 5 min, and then dried by centrifugation at 600 rpm for 5 min.

The microarrays were scanned at 70% photomultiplier tube and 100% laser power settings using a Genepix 4000B confocal scanner (Axon Instruments, Sunnyvale, CA). Genepix 3.0 software (Axon Instruments) was used to analyze the acquired images at 532 nm for Cy3 detection. The average background was subtracted and the values < average background ±2 standard deviation (SD) were removed from each data point. Median of the effective data points of each lectin was globally normalized to the sum of medians of all effective data points for each lectin in one block. Each sample was observed consistently by three repeated slides and the normalized medians of each lectin from nine repeated blocks were averaged and the SD was determined. The normalized data of each sample were compared with each other based upon fold-changes according to the following criteria: fold changes >2 or <0.5 indicated an up-regulation or a down-regulation, respectively. Differences between the two arbitrary data sets or multiple data sets were tested by Student's t test or one-way ANOVA to each lectin signal using SPSS v. 19. The original data was further analyzed by Expander 6.0 (http://acgt.cs.tau.ac.il/expander/) in order to perform a hierarchical clustering analysis.

The pooled sera of the HV, CHB, and CHC patients were analyzed by SDS-PAGE and subsequently lectin blotting (Blomme et al., 2009). For SDS-PAGE, samples were boiled for 5 min at 100°C mixed with 5 × loading buffer, and run on a 10% polyacrylamide resolving gel and a 5% stacking gel. Molecular mass standards (Thermo Scientific, Waltham, MA) were run with all gels. Some gels were then stained directly with alkaline silver. For lectin blotting, the proteins in gels were then transferred to a PVDF membrane (Immobilon-P; Millipore, Bedford, MA) with a semi-dry transfer unit (Hoefer Scientific, Holliston, MA) for 1.5 h at 24 mA. The membranes were washed twice with TTBS (150 mM NaCl, 10 mM Tris-HCl, 0.05% v/v Tween 20, pH 7.5) and then blocked for 1 h with Carbo-Free Blocking Solution (Vector, Burlingame, CA) at room temperature. On the basis of silver staining of the gels after transfer, it was evident that lower-molecular-mass proteins (<50 kDa) transferred more thoroughly to blots. Hence, if any bias was present in final results, it favored the detection of proteins smaller than 50 kDa during blotting with lectin. The membranes were then washed again and incubated with Cy5 (GE Healthcare) labeled lectins (2 μg/mL in Carbo-Free blocking solution) with gentle shaking overnight at 4°C in the dark. The membranes were washed twice each with TTBS for 10 min and scanned by red fluorescence channel (635 nm excitation/650LP emission) with the voltage of 800 PMT using a phosphorimager (Storm 840, Molecular Dynamics, Sunnyvale, CA).

The lectin-magnetic particle conjugate-based method was utilized to selectively isolate the glycoproteins according to our previous protocol (Yang et al., 2012, 2013). Briefly, 2 mg of the pooled serum proteins from the HV, CHB and CHC patients were diluted to 600 μL with the binding buffer (0.1 M Tris-HCl, 0.15 M NaCl, 1 mM CaCl₂, 1 mM MgCl₂, and 1 mM MnCl₂, pH 7.4) supplemented with 6 μL proteinase inhibitor cocktail. The homemade PHA-E+L-magnetic particles were rinsed three times using the binding buffer, followed by incubation with the diluted sera at room temperature for 1 h under gentle shaking. After incubation, the unbound proteins were removed by washing three times with the washing buffer (the binding buffer supplemented with 0.1% Tween 20, pH 7.2) thoroughly. The glycoproteins bound to the PHA-E+L-magnetic particles were eluted with 500 μL of the elution buffer (8 M urea in 0.1 M NH₄HCO₃) at room temperature for 1 h under gentle shaking.

The N-linked glycans were released from the isolated proteins with PNGase F according to our previous protocol with some modifications (Yang et al., 2012, 2013). The glycoproteins were concentrated and desalted by adding to a size-exclusion spin ultrafiltration (Amicon Ultra-0.5 10 K device, Millipore) with a molecular mass cutoff of 10 kDa. The obtained glycoproteins were denatured with 8 M urea, 10 mM DTT (Sigma-Aldrich), 10 mM IAM (Sigma-Aldrich), and 100 μL NH₄HCO₃ solutions (50 mM, pH 8.0). The ultrafiltration unit was centrifuged at 12,000 × g for 15 min followed by adding 10 μL of PNGase F (New England BioLabs, Ipswich, MA). Incubation was porformed at 37°C overnight to release the N-linked glycans from the glycoproteins. The reaction was stopped by incubating the mixture at 80°C for 5 min. The glycans were collected by centrifugation with 200 μL 40 mmol/L NH₄HCO₃. Then, the N-glycans were purified using Sepharose 4B (Sigma-Aldrich). Sepharose 4B in a microtube was washed with methanol/water (1:1, v/v) and 1-butanol/methanol/water (5:1:1, v/v) under centrifugation. Glycans were dissolved in 500 μL 1-butanol/methanol/water (5:1:1, v/v) and added to the microtube. The mixture was gently shaken for 45 min and washed three times with 1-butanol/methanol/water (5:1:1, v/v). N-glycans were eluted with methanol/water (1:1, v/v), and the eluent was collected and lyophilized.

The glycan mixture was dissolved in 10 μL of 50% v/v methanol, and 1 μL were spotted directly on an MTP AnchorChip var/384 sample target and dried. Then an equal volume 20 mg/mL DHB in 50% v/v methanol solution was spotted to re-crystallize the glycans. The target was introduced in a mass spectrometer MALDI-TOF/TOF (UltrafleXtreme, Bruker Daltonics, Bremen, Germany). Ionization was performed in MS and MS/MS by irradiation of a nitrogen laser (337 nm) operating at 1 kHz. Data were acquired at a maximum accelerating potential of 25 kV in the positive and reflectron modes. Mass calibration was done using the peptide calibration standards 250 calibration points from Bruker Daltonics. A total of 1,500 laser shots per pixel (200 laser shots per position of a random walk within each pixel) were collected and the data were acquired using the Flex software suite (FlexControl3.3, Flex-Analysis 3.3). The data of m/z were analyzed and annotated by Glycoworkbench software (Ceroni et al., 2008). By combining the information from glycan structures recognized by a lectin and received from fragmentation of [M+H]⁺ and [M+Na]⁺ ions of the glycans, a complete structural characterization in terms of linkage, branching of glycosidic bonds of oligosaccharides were achieved.

A lectin microarray format and the glycopatterns of Cy3-labeled pooled sera from the HV, CHB, and CHC patients bound to the lectin microarrays are shown in Figures 1A,B. The averaged normalized fluorescent intensities (NFIs) of each lectin from the HV, CHB, and CHC patients are summarized as the mean values ± SD in Table S1. The generated data from three biological replicates were imported into EXPANDER 6.0 to perform a hierarchical clustering analysis (Figure 1C). The NFIs of each lectin from the HV, CHB, and CHC patients were compared each other based on fold change in pairs (fold change >2.0 or <0.5 fold), with all p-values below 0.05, to evaluate whether the glycopatterns of serum proteins were altered between them (Table 2). The results showed that there were 19 lectins to exhibit significant difference associated with virus-infected hepatitis in serum glycopatterns compared with the HV. Among the total, there were 11 lectins (e.g., the High-Man and Manα1-3Man binder HHL, GlcNAc and agalactosylated tri/tetra antennary glycans binder GSL-II, and Galα1-3(Fucα1-2)Gal binder EEL) that exhibited significantly increased NFIs (all fold changes ≥ 2.19, p ≤ 0.05), and 3 lectins (the Fucα-1,6GlcNAc (core fucosylated) binder LCA, and Fucα-1,6GlcNAc binder ACA as well as GalNAcα-Ser/Thr(Tn) and GalNAcα1-3Gal binder VVA) exhibited significantly decreased NFIs (all fold changes ≤ 0.48, p ≤ 0.002) only in the CHB patients (Figure 1D). However, The GalNAcα1-3(Fucα1-2)Gal binder DBA exhibited significantly decreased NFIs (fold change = 0.47, p ≤ 0.004), the multivalent Sia and (GlcNAc)_n binder WGA, and Sia2-6Gal/GalNAc binder SNA exhibited significantly increased NFIs (all fold changes ≥ 2.00, p ≤ 0.05) only in the CHC patients. And the (GlcNAc)n and high mannose-type N-glycans binder LEL, and Galβ1-4/3GlcNAc binder MAL-I showed a coincidentally increasing trend in both CHC and CHB patients compared with the HV (all fold changes ≥ 2.97, p ≤ 0.058) (Figure 1E).

To evaluate the reliability of the serum protein glycopatterns of the HV, CHB, and CHC patients from the results of the lectin microarrays, an additional cohort of 15 HV, 22 CHB, and 23 CHC patients was selected randomly and tested one by one using the same lectin microarrays. The results of individual analysis also demonstrated that there were eight lectins (MPL, GSL-I, PTL-II, UEA-I, WGA, LEL, VVA, and MAL-I) exhibited a high degree of consistency with the pooled samples of HV, CHB, and CHC patients. Besides, a bisecting GlcNAc, bi-antennary N-glycans, tri-, and tetra-antennary complex-type N-glycans binder PHA-E+L exhibited significantly decreased NFIs in the CHB compared with HV and CHC subjects (p < 0.01) (Figure 2). Notably, PHA-E+L were also associated with the decreased NFIs in the pooled serum samples of CHB compared with that of HV and CHC subjects (all fold changes ≤ 0.69, p < 0.001).

Lectin blotting analyses (GSL-I, LEL, and PHA-E+L) were performed to investigate the difference of pooled serum glycopattern abundances between HV, CHB, and CHC patients (Figure 3A). The results of SDS-PAGE demonstrated that their serum protein bands were similar, the highest abundant bands with molecular weights (Mr) of 55–70 kDa is albumin. The result of the lectin blotting analysis showed the different apparent bands range from 10 to 250 kDa. The αGalNAc, αGal, and GalNAcα-Ser/Thr (Tn) binder GSL-I and the high mannose-type N-glycans binder LEL showed strong binding to three bands (b4, b5, b8) in the CHB patients than in the HV and CHC patients. On the contrary, PHA-E+L exhibited a weaker binding to mainly b5 band in the CHB patients compared with the HV and CHC patients. The sum fluorescence signal intensity of the bands for all subject groups was performed by Image J (Figure 3B). The results were coincident with the results from the lectin microarrays.

To obtain the bi-, tri-, and tetra-antennary complex-type N-glycan structures of glycoproteins in sera from the HV, CHB, and CHC patients, the glycoproteins were isolated using the PHA-E+L-magnetic particle conjugates, then N-glycans were released from the isolated glycoproteins by PNGase F, and characterized by MALDI-TOF/TOF-MS, respectively. Representative MS spectra of N-glycans with signal-to-noise ratios >6 were annotated using the GlycoWorkbench software. As a result, the MALDI-TOF/TOF-MS peaks of N-linked glycans from the isolated glycoproteins in sera of the HV, CHB, and CHC patients were shown in Figures 4A–C. There were 18 N-glycan peaks (e.g., m/z 1663.734, 1809.849, and 1921.809), 23 N-glycan peaks (e.g., m/z 1419.743, 1663.734, and 1743.581) and 26 N-glycan peaks (e.g., m/z 1866.661, 2012.908, and 2039.542) to be identified in HV, CHB, and CHC patients, respectively, and their proposed structures were listed in Table S2. Of these N-linked glycans, it was noticed that there was an overlap of 23 N-glycan peaks (e.g., m/z 1419.743, 1663.734, and 1743.581) between CHB, and CHC patients, 5 glycan peaks (e.g., m/z 1850.878, 1866.661, and 2037.750) were presented in virus-infected hepatitis patients compared with HV, 3 glycan peaks (e.g., 1460.659, 2069.740, and 2174.772) were observed only in CHC patients.

To provide insight into the substitution and branching pattern of the monosaccharide constituents, the majority of the glycan peaks observed in the CHC MS spectrum were assigned to tandem MS analysis. The MS/MS spectra of the precursor ions m/z 1905.634 and 2288.840 were illustrated in Figure 5. The results showed that the B-, C-, and Y-type cleavage that provided detailed sequence and branching information were the major fragment ion due to cleavage needed low energy. The cross-ring fragment ions that provide composition and linkage information were also detected. For instance, the fragment ions B₂ (388.121), B₃ (550.174), ^{2, 4}A_GlcNAcY₃ (998.344), and C^{2, 4}X_GlcNAc (1420.497) in the m/z 1905.634 elucidated the condition of the triantennary N-glycans and the occurrence of the terminal β1,4Gal. Fragment ions C₂ (692.248), C₁Y₃ (714.243), B32,4X_Fuc (782.269), C^{1, 5}X_GlcNAc (1561.539), and ^{1, 5}A_GlcNAcY₃ (1838.655) in the m/z 2288.840 indicated that the existence of both core and terminal fucose. In all, this method did not only demonstrate the overall difference in glycosylation, but also could reflect the differences between individual structures.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.