Site-specific glycosylation of serum antithrombin-III as a potential biomarker for AFP-negative HCC.

Early diagnosis of hepatocellular carcinoma (HCC), particularly in alpha-fetoprotein (AFP)-negative patients, remains a significant challenge. Characterizing N-glycopeptides with site-specific glycan structural information enables a better understanding of the molecular pathogenesis of liver injury and cancer. Here, we performed an unbiased quantitative analysis of N-glycopeptides from serum in patients with hepatitis B virus (HBV)-related liver diseases using a stable isotope labeling-based glycoproteomic approach. Serum samples from patients with liver cirrhosis (LC) and AFP-negative HCC were compared. A total of 264 unique N-glycopeptides were initially identified, with 30 high-confidence intact glycopeptides retained after stringent quality control. Following immunoprecipitation of ATIII, comparative analysis confirmed a significant downregulation of two ATIII glycopeptides carrying a biantennary disialylated (H5N4S2) glycan at asparagine residues N128 and N224 in AFP-negative HCC compared with LC (p < 0.01). A small AFP-positive HCC group was examined as an exploratory reference. Occupancy of four N-glycosylation sites (N128, N167, N187, N224) was verified, and site-specific glycan heterogeneity was delineated. These findings suggest that site-specific glycosylation changes on ATIII may serve as candidate biomarkers to complement current diagnostic strategies for AFP-negative HCC.