Shirai Atsuko, Matsuyama Akihisa, Yashiroda Yoko, Hashimoto Atsushi, Kawamura Yumi, Arai Ritsuko, Komatsu Yasuhiko, Horinouchi Sueharu, Yoshida Minoru
Chemical Genetics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
J Biol Chem. 2008 Apr 18;283(16):10745-52. doi: 10.1074/jbc.M709211200. Epub 2008 Feb 21.
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.
SDS - PAGE是一种长期以来用于根据蛋白质分子大小分离蛋白质的基本方法。尽管其操作简单，但它能提供有关蛋白质特性的信息，这些信息超出了其分子量范围，因为蛋白质的凝胶迁移率常常反映其物理化学性质和翻译后修饰。在此，我们报告了对蛋白质组凝胶迁移率的全面分析，我们将其称为“迁移组”，该分析涵盖了裂殖酵母蛋白质组的93.4%。令我们惊讶的是，超过40%的蛋白质并未迁移到其计算位置。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。这个具有高真实迁移率覆盖率的实验数据集，使得在不使用任何专门技术的情况下识别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到蛋白质先前未知的翻译后修饰；例如，我们发现eIF5A是一种与Sir2相关的脱乙酰酶Hst2的新底物。此外，我们使用特异性抗乙酰化和抗甲基化赖氨酸抗体同时鉴定出了12种乙酰化蛋白质和8种甲基化蛋白质，其中大多数此前未知会发生这些修饰。因此，我们提出迁移组和基于电泳的方法对于鉴定和表征凝胶上检测到的蛋白质具有普遍实用性。
