Cao Xinyi, Cao Zhao, Shao Yuyin, Liu Chao, Yan Guoquan, Meng Xinmin, Zhang Lei, Chen Chen, Huang Guiyue, Shu Hong, Lu Haojie
Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
Department of Clinical Laboratory, First Affiliated Hospital of Guangxi Medical University, Nanning, China.
Front Oncol. 2021 Apr 6;11:651421. doi: 10.3389/fonc.2021.651421. eCollection 2021.
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 O/O 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 HANWTLTPLK (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.
甲胎蛋白（AFP）阴性肝细胞癌（HCC）的诊断主要依赖于影像学和病理学检查，且缺乏有价值的实用标志物。蛋白质N-糖基化是一种关键的翻译后修饰过程，与生物体中的许多生物学功能相关。N-糖基化的改变与包括肝细胞癌在内的炎症性疾病和感染性疾病相关。在此，使用无标记定量方法，在一个包括AFP阴性HCC和肝硬化（LC）患者的发现队列（n = 40）中分析了分子量（MW）为40 - 55 kDa的血清N-连接完整糖肽。进一步采用定量刀豆球蛋白A（LCA）ELISA法确认肝病患者（n = 56）血清对氧磷酶1（PON1）糖基化的差异。然后，在一个验证队列（n = 64）中，通过免疫沉淀（IP）和基于质谱的O/O C末端标记定量方法全面评估PON1位点特异性完整N-糖肽的改变，以区分AFP阴性HCC患者和LC患者。使用专用搜索引擎pGlyco共鉴定出195种糖肽。其中，与LC相比，AFP阴性HCC中来自载脂蛋白H（APOH）、肝促凝血酶原激酶/肝促凝血酶原激酶受体（HPT/HPTR）和PON1的糖肽有显著变化。此外，AFP阴性HCC患者中PON1与LCA的反应性显著高于肝硬化患者。与LC患者相比，AFP阴性HCC患者中与PON1对应的两种糖肽HANWTLTPLK（H5N4S2）和（H5N4S1）显著增加。PON1糖基化的变化可能与AFP阴性HCC相关，并且可能有助于作为潜在的基于糖组学的生物标志物来区分AFP阴性HCC和肝硬化。
