Identification and Confirmation of Biomarkers Using an Integrated Platform for Quantitative Analysis of Glycoproteins and Their Glycosylations

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. However, accurate diagnosis can be difficult as most of the patients who develop this tumor have symptoms similar to those caused by longstanding liver disease. Herein we developed an integrated platform to discover the glycoprotein biomarkers in early HCC. At first, lectin arrays were applied to investigate the differences in glycan structures on serum glycoproteins from HCC and cirrhosis patients. The intensity for AAL and LCA was significantly higher in HCC, indicating an elevation of fucosylation level. Then serum from 10 HCC samples and 10 cirrhosis samples were used to screen the altered fucosylated proteins by a combination of Exactag labeling, lectin extraction and LC-MS/MS. Finally, 27 HCC and 27 cirrhosis serum samples were used for lectin-antibody arrays to confirm the change of these fucosylated proteins. C3, CE, HRG, CD14 and HGF were found to be biomarker candidates for distinguishing early HCC from cirrhosis, with a sensitivity of 72% and specificity of 79%. Our work gives insight to the detection of early HCC, and the application of this comprehensive strategy has the potential to facilitate biomarker discovery on a large scale.

肝细胞癌（HCC）是肝脏最常见的原发性恶性肿瘤。然而，由于大多数患有此肿瘤的患者症状与长期肝病的症状相似，因此准确诊断颇具挑战。本研究开发了一个综合平台以发现早期肝细胞癌的糖蛋白生物标志物。起初，通过应用凝集素阵列来探究肝细胞癌和肝硬化患者血清糖蛋白上糖苷结构差异。AAL和LCA的强度在肝细胞癌中显著升高，表明岩藻糖基化水平上升。随后，使用10份肝细胞癌样本和10份肝硬化样本的血清，通过Exactag标记、凝集素提取和液相色谱-质谱-质谱联用（LC-MS/MS）的组合筛选出改变的岩藻糖基化蛋白。最终，使用27份肝细胞癌和27份肝硬化血清样本进行凝集素-抗体阵列以确认这些岩藻糖基化蛋白的变化。C3、CE、HRG、CD14和HGF被发现是区分早期肝细胞癌与肝硬化的生物标志物候选，其敏感性为72%，特异性为79%。本研究为早期肝细胞癌的检测提供了洞见，并且该全面策略的应用具有在大规模上促进生物标志物发现的可能性。