Global analysis of gel mobility of proteins and its use in target identification
Global analysis of gel mobility of proteins and its use in target identification
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DOI：
10.1074/jbc.m709211200
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发表时间：
2008-04-18
影响因子：
4.8
通讯作者：
Yoshida, Minoru
中科院分区：
文献类型：
--
作者：
Shirai, Atsuko;Matsuyama, Akihisa;Yoshida, Minoru
SDS-PAGE是一种基本方法，长期以来一直用于根据分子大小分离蛋白质。尽管它的简单性，它提供了信息的蛋白质的特征超出其分子量，因为蛋白质的凝胶迁移率往往反映了他们的物理化学性质和翻译后修饰。在这里，我们报告的蛋白质组，我们称之为“流动组”，覆盖93.4%的裂变酵母蛋白质组的凝胶流动性的全球分析。令我们惊讶的是，超过40%的蛋白质没有迁移到它们的计算位置。统计分析表明，这种差异在很大程度上取决于蛋白质的疏水性。该实验数据集具有真实的迁移率的高覆盖率，使得在不使用任何专门技术的情况下鉴定凝胶上检测到的蛋白质成为可能。这种方法使我们能够检测以前未知的蛋白质翻译后修饰;例如，我们发现eIF 5A是Sir 2相关的脱乙酰酶Hst 2的新底物。此外，我们同时确定了12个乙酰化和8个甲基化的蛋白质使用特定的抗乙酰化和抗甲基化赖氨酸抗体，其中大部分还没有被称为是受修改。因此，我们提出了一般的实用性的mobilitome和基于荧光素酶的方法，用于鉴定和表征的蛋白质上检测到的凝胶。
SDS-PAGE is a basic method that has long been used for separation of proteins according to their molecular sizes. Despite its simplicity, it provides information on characteristics of proteins beyond their molecular masses because gel mobility of proteins often reflects their physicochemical properties and post-translational modifications. Here we report on a global analysis of gel mobility of the proteome, which we term the "mobilitome," covering 93.4% of the fission yeast proteome. To our surprise, more than 40% of proteins did not migrate to their calculated positions. Statistical analyses revealed that the discrepancy was largely dependent on the hydrophobicity of proteins. This experimental data set, with a high coverage rate of real mobility, made it feasible to identify proteins detected on the gel without using any specialized techniques. This approach enabled us to detect previously unknown post-translational modifications of a protein; for example, we revealed that eIF5A is novel substrate of a Sir2-related deacetylase Hst2. Furthermore, we concomitantly identified twelve acetylated and eight methylated proteins using specific anti-acetylated and anti-methylated lysine antibodies, most of which had not been known to be subject to the modifications. Thus, we propose the general usefulness of the mobilitome and electrophoresis-based methodology for the identification and characterization of proteins detected on the gel.