A Panel of Glycopeptides as Candidate Biomarkers for Early Diagnosis of NASH Hepatocellular Carcinoma Using a Stepped HCD Method and PRM Evaluation
Yu Lin, Jianhui Zhu, Lingyun Pan, Jie Zhang, Zhijing Tan, Jocelyn Olivares, Amit G. Singal, Neehar D. Parikh, David M. Lubman
Abstract
Changes in N-glycosylation on specific peptide sites of serum proteins have been investigated as potential markers for diagnosis of nonalcoholic steatohepatitis (NASH)-related HCC. To accomplish this work, a novel workflow involving broad-scale marker discovery in serum followed by targeted marker evaluation of these glycopeptides were combined. The workflow involved an LC-Stepped HCD-DDA-MS/MS method coupled with offline peptide fractionation for large-scale identification of N-glycopeptides directly from pooled serum samples (each n = 10) as well as differential determination of N-glycosylation changes between disease states. We then evaluated several potentially diagnostic N-glycopeptides among 78 individual patient samples (40 cirrhosis, 28 early stage NASH HCC, and 10 late-stage NASH HCC) by LC-Stepped HCD-PRM-MS/MS to quantitatively analyze 65 targeted glycopeptides from 7 glycoproteins. Of these targets, we found site-specific N-glycopeptides n169GSLFAFR_HexNAc(4)Hex(5)NeuAc(2) and n242ISDGFDGIPDNVDAALALPAHSYSGR_HexNAc(5)Hex(6)Fuc(1)NeuAc(3) from VTNC were significantly increased comparing samples from patients with NASH cirrhosis and NASH HCC (p < 0.05). When combining results of these 2 glycopeptides with AFP, the ROC curve analysis demonstrated the AUC value increased to 0.834 (95% CI, 0.748–0.921) and 0.847 (95% CI, 0.766–0.932), respectively, as compared to that of AFP alone (AUC = 0.791, 95% CI, 0.690–0.892). These 2 glycopeptides may serve as potential biomarkers for early HCC diagnosis in patients with NASH related cirrhosis.