蛋白质组学技术分析肝癌细胞HCCLM3的细胞外蛋白降解组

Degradomic analysis of extracellular proteins of hepatocellular carcinoma cell line hcclm3 using proteomic approach

目的:分析肝癌细胞系HCCLM3的细胞外蛋白降解组,筛选肝癌标记物。方法:凝胶电泳分离HCCLM3细胞外蛋白,线性离子阱-傅立叶变换离子回旋共振质谱(LTQ-FT-MS/MS)对其鉴定,数据库检索结合分子质量分析其降解组。免疫杂交定量骨桥蛋白(OPN)在正常、乙肝、乙肝肝硬化和肝癌4组血清中的表达。结果:HCCLM3细胞外蛋白电泳共测得51个条带,鉴定出1 405种非冗余蛋白,其中474种蛋白发生降解,且分泌蛋白的降解率最高(50%)。肝癌患者血清中OPN 20 kD片段明显高于其余3组(P<0.05),受试者工作特征曲线(ROC)显示AFP对肝癌诊断的曲线下面积(AUC)为0.79,OPN 20 kD片段的AUC为0.68,两者结合后能提高AUC至0.89。结论:凝胶电泳结合质谱技术研究肝癌蛋白降解组有效可行,骨桥蛋白20 kD片段可能对肝癌的诊断具有价值。

Objective： To analyze the degradome of extracellular proteins of hepatocellular carcinoma（HCC） cell line HCCLM3 through proteomic techniques,so as to screen a marker for HCC. Methods： Extracellular proteins of HCCLM3 were resolved by polyacylamide gel electrophoresis（PAGE）,and were then identified by using Linear Ion Trap Quadrupole-Fourier Transform-Mass Spectrometry（LTQ-FT-MS/MS） after in-gel digestion.The degradome was analyzed through on-line database searching combined with molecular weight information.The osteopontin（OPN） expression in the plasma of healthy subjects,hepatitis B,hepatitis B-caused cirrhosis and HCC patients were quantified by Western blot method. Results： The HCCLM3 extracellular proteins were resolved into 51 bands and 1 405 non-redundant proteins were identified,Among which 474 proteins were degradated,and the secretory proteins had the highest degradation ratio（50%）.The serum level of 20-kD fragment of OPN was significantly higher in HCC patients than that in other three populations.The receiver operating characteristic（ROC） showed the area under curve（AUC） of AFP for HCC diagnosis was 0.79 and 20-kD fragment of OPN was 0.68,respectively,and the combination of the two could raise the AUC to 0.89. Conclusion： The combination use of gel electrophoresis and MS is an effective and feasible approach to investigate the HCC degradome.The 20-kD fragment of OPN may be a potential marker for HCC diagnosis.