Glycopeptides with Sialyl Lewis Antigen in Serum Haptoglobin as Candidate Biomarkers for Nonalcoholic Steatohepatitis Hepatocellular Carcinoma Using a Higher-Energy Collision-Induced Dissociation Parallel Reaction Monitoring-Mass Spectrometry Method

[Image: see text] Nonalcoholic steatohepatitis (NASH) is the fastest growing cause of hepatocellular carcinoma (HCC) in the United States. Changes in N-glycosylation on specific glycosites of serum proteins have been investigated as potential markers for the early detection of NASH-related HCC. Herein, we report a glycopeptide with a Sialyl Lewis structure derived from serum haptoglobin (Hp) as a potential marker for NASH related HCCs among 95 patients with NASH, including 46 cirrhosis, 32 early-stage HCC, and 17 late-stage HCC. Hp immuno-isolated from patient serum was analyzed using LC-HCD-PRM-MS/MS followed by data analysis via Skyline software. Two glycopeptides involving site N184 and four glycopeptides involving site N241 were significantly changed in patients with HCC vs NASH cirrhosis (P < 0.05). The two-marker panel using N-glycopeptide N241_A4G4F2S4 showed the best performance for HCC detection when combined with α-fetoprotein (AFP), with an improved estimated area under the curve (AUC) = 0.898 (95% CI: 0.835, 0.951), compared to the AUC of 0.790(95% CI, 0.697 0.872) using AFP alone (P = 0.048). At 90% specificity, the combination of N241_A4G4F2S4 + AFP had an improved sensitivity of 63.3%, compared to the sensitivity of 52.3% using AFP alone. When using three markers, the panel of AFP + N241_A2G2F1S2 + N241_A4G4F2S4 yielded an estimated AUC of 0.928 (95% CI: 0.877, 0.970). Our findings indicated that N241_A4G4F2S4 may play an important role in distinguishing HCC from NASH cirrhosis.