肝癌具有发病率高和早期诊断难的特点，建立灵敏特异的准确和完整识别糖蛋白蛋白表达量、糖链结构、糖基化修饰程度和糖基化修饰位点变化的新方法，对于肝癌的早期诊断具有重要意义。我们前期研究发现一组分子量40kDa左右的糖蛋白在肝癌中聚糖变化差异大，很可能是肝硬化发展到肝癌的重要早期诊断标志物群。本项目拟建立基于同位素标记的完整N-糖肽的微观多样性的质谱定量解析新方法，同步定量肝硬化和肝癌患者血清40kDa糖蛋白分子群的完整糖肽和非糖肽，可获得完整糖肽的微观多样性的轮廓识别模式。该新方法以这群糖蛋白自身的非糖肽作为内标，可消除蛋白自身含量变化对糖链微观不均一性的影响；并且在此基础上，完整糖肽轮廓识别模式又以各完整糖肽互为内标，可进一步提高40kDa糖蛋白分子群作为标志物识别的准确性。将精细的N-糖链微观差异表达应用于肝癌的预测诊断中，不仅弥补单一指标检测的不足，更为肝癌的糖标志物研究提供崭新的思路。

Hepatocellular carcinoma (HCC) is the major primary liver cancer with high mortality and the early diagnosis for HCC is far from satisfactory. Novel mass spectrometry approaches for sensitive and accurate assessment of protein expression, glycan structure, glycosylation level and glycosite of glycoprotein are important, especially for early diagnosis of HCC. The glycan changes of group of sera 40kDa glycoproteins were found in HCC and liver cirrhosis (LC) in our previous study. This group may be the potential glycobiomarker for distinguishing early HCC from LC patients. In this study, a novel strategy based on isotope labeling of intact N-glycopeptide is developed to quantify glycopeptide and non-glycosylated peptides of this goup simultaneously, and obtain pattern recognition of intact glycopeptide microheterogeneity. The non-glycosylated peptides are used as internal standards to obtain site-specific microheterogeneity level on its protein level and the glycopeptides are also used as internal standards to improve accuracy. The pattern recognition of intact glycopeptide of this group of 40kDa glycoproteins may be a more powerful tool to aid diagnosis of HCC than single biomarker.