Abstract
The diagnosis of AFP (alpha-fetoprotein)-negative HCC (hepatocellular carcinoma) mostly relies on imaging and pathological examinations, and it lacks valuable and practical markers. Protein N-glycosylation is a crucial post-translation modifying process related to many biological functions in an organism. Alteration of N-glycosylation correlates with inflammatory diseases and infectious diseases including hepatocellular carcinoma. Here, serum N-linked intact glycopeptides with molecular weight (MW) of 40-55 kDa were analyzed in a discovery set (n = 40) including AFP-negative HCC and liver cirrhosis (LC) patients using label-free quantification methodology. Quantitative lens culinaris agglutin (LCA) ELISA was further used to confirm the difference of glycosylation on serum PON1 in liver diseases (n = 56). Then, the alteration of site-specific intact N-glycopeptides of PON1 was comprehensively assessed by using Immunoprecipitation (IP) and mass spectrometry based 16O/18O C-terminal labeling quantification method to distinguish AFP-negative HCC from LC patients in a validation set (n = 64). Totally 195 glycopeptides were identified using a dedicated search engine pGlyco. Among them, glycopeptides from APOH, HPT/HPTR, and PON1 were significantly changed in AFP-negative HCC as compared to LC. In addition, the reactivity of PON1 with LCA in HCC patients with negative AFP was significantly elevated than that in cirrhosis patients. The two glycopeptides HAN253WTLTPLK (H5N4S2) and (H5N4S1) corresponding to PON1 were significantly increased in AFP-negative HCC patients, as compared with LC patients. Variations in PON1 glycosylation may be associated with AFP-negative HCC and might be helpful to serve as potential glycomic-based biomarkers to distinguish AFP-negative HCC from cirrhosis.