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目的:应用蛋白质组学双向荧光差异凝胶电泳和质谱技术筛选肺癌骨转移血清标志。方法:收集肺癌伴骨转移、无骨转移各24例血清样本。同组血清等量混合,用除白蛋白试剂盒除去血清中的白蛋白,再经除盐浓缩后,将4组处理后的血清样品用不同的CyDye染料交叉标记后进行双向荧光差异凝胶电泳(2-DDIGE)。利用DeCyder软件分析,将肺癌伴骨转移组与肺癌无骨转移组比较的差异蛋白质点行基质辅助激光解析离子化飞行时间质谱(MALDI-TOF-MS)鉴定。结果:双向荧光差异凝胶电泳显示,肺癌骨转移组血清蛋白中有16个斑点,与肺癌无骨转移组相比差异有统计学意义。其中11个蛋白质表达水平显著增加,选取5个蛋白质表达水平显著下降。5个差异蛋白点进行胶内原位酶解、肽质指纹图谱分析,成功得到4个蛋白质肽质指纹图,并查询数据库初步鉴定该4个蛋白质分别为视黄醇结合蛋白-甲状腺素运载蛋白复合物(RBP-TTR)、结合珠蛋白(Hp)、S-亚硝基血红蛋白(SNO-Hb)和de-sArg141αHb。结论:双向荧光差异凝胶电泳结合质谱鉴定是血清差异蛋白质组学研究的可靠平台和有力工具。所鉴定出的蛋白质可能与肺癌骨转移的发生、发展有关,有助于肺癌骨转移发病机制的了解。
Objective: To screen serum markers of bone metastasis of lung cancer by proteomic two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. Methods: 24 cases of lung cancer with bone metastasis and no bone metastasis were collected. Serum equal to the same group were mixed with albumin in serum albumin removed, and then desalted concentration, the four groups of serum samples after treatment with different CyDye dye cross-labeling after two-way fluorescence difference gel electrophoresis (2-DDIGE). DeCyder software was used to identify the differentially expressed proteins of lung cancer with bone metastasis and non-bone metastasis of lung cancer by MALDI-TOF-MS. Results: Two-dimensional fluorescence difference gel electrophoresis showed that there were 16 spots in the serum protein of lung cancer bone metastasis group, which was significantly different from that of non-bone metastasis lung cancer group. Among them, the expression of 11 proteins increased significantly, and the expression level of 5 proteins decreased significantly. Five differential protein spots were subjected to in-gel enzymatic digestion and peptide mass fingerprinting analysis. Four protein peptide fingerprints were successfully obtained, and the four proteins were initially identified as retinol binding protein-transthyretin Complex (RBP-TTR), haptoglobin (Hp), S-nitrosohemoglobin (SNO-Hb) and de-sArg141αHb. Conclusion: Two-dimensional fluorescence differential gel electrophoresis combined with mass spectrometry identification is a reliable platform and a powerful tool for the differential proteomics study of serum. The identified proteins may be related to the occurrence and development of bone metastasis of lung cancer and contribute to the understanding of the pathogenesis of bone metastasis of lung cancer.