Identification of altered protein expression and post-translational modifications in primary colorectal cancer by using agarose two-dimensional gel electrophoresis
Identification of altered protein expression and post-translational modifications in primary colorectal cancer by using agarose two-dimensional gel electrophoresis
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DOI：
10.1158/1078-0432.ccr-03-0321
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发表时间：
2004-03-15
影响因子：
11.5
通讯作者：
Nomura, F
中科院分区：
文献类型：
--
作者：
Tomonaga, T;Matsushita, K;Nomura, F
目的：尽管最近进行了大量的蛋白质组学研究，以确定蛋白质表达的癌症相关变化，但仅确定了相对丰富的蛋白质的有限展示。本研究的目的是利用高分辨率二维凝胶电泳（2-DE）鉴定原发性结直肠癌组织中作为潜在肿瘤标志物的新蛋白。实验设计：采用琼脂糖凝胶等电聚焦2-DE法比较10例结直肠癌组织与邻近正常粘膜的蛋白谱。采用Western blot分析和免疫组织化学检测几种蛋白的表达改变和翻译后修饰。结果：用质谱法鉴定了正常组织与肿瘤组织匹配差异表达的107个位点的97个蛋白（90.7%）。其中42个特异蛋白（49个点）在肿瘤中显著升高或降低。它们包括真核翻译起始因子411、无机焦磷酸酶、前梯度2同源物、醛缩酶A和细胞内氯离子通道1，经Western blot分析和免疫组织化学证实其在肿瘤组织中的表达升高。有趣的是，在大多数肿瘤组织中，真核翻译起始因子4H的两种转录变体中只有异构体1被大幅上调。此外，还鉴定了脯氨酸-4-羟化酶β亚基和膜联蛋白A2的翻译后修饰。结论：我们使用琼脂糖2-DE鉴定了几种在原发性结直肠癌中表达改变的新蛋白。这种方法是一种强大的技术，它不仅可以搜索感兴趣的生物过程中的定量变化，还可以搜索质变，并可能有助于更深入地了解人类癌症的潜在机制。
Purpose: Although numerous proteome studies have been performed recently to identify cancer-related changes in protein expression, only a limited display of relatively abundant proteins has been identified. The aim of this study is to identify novel proteins as potential tumor markers in primary colorectal cancer tissues using a high-resolution two-dimensional gel electrophoresis (2-DE).Experimental Design: 2-DE using an agarose gel for isoelectric focusing was used to compare protein profiling of 10 colorectal cancer tissues and adjacent normal mucosa. Altered expression and post-translational modification of several proteins were examined using Western blot analysis and inummohistochemistry.Results: Ninety-seven proteins of 107 spots (90.7%) that were differentially expressed between matched normal and tumor tissues were identified by mass spectrometry. Among them, 42 unique proteins (49 spots) significantly increased or decreased in the tumors. They include eukaryotic translation initiation factor 411, inorganic pyrophosphatase, anterior gradient 2 homologue, aldolase A, and chloride intracellular channel 1, whose elevated expression in tumor tissues was confirmed by Western blot analysis and immunohistochemistry. Interestingly, only isoform 1 of two transcript variants of eukaryotic translation initiation factor 4H was greatly up-regulated in most of the tumor tissues. Moreover, post-translational modifications of the prolyl-4-hydroxylase beta subunit and annexin A2 also were identified.Conclusions: We identified several novel proteins with altered expression in primary colorectal cancer using agarose 2-DE. This method is a powerful technique with which to search for not only quantitative but also qualitative changes in a biological process of interest and may contribute to the deeper understanding of underlying mechanisms of human cancer.