Post-translational modifications to tubulin such as acetylation and detyrosination play important roles in microtubule functions. Methylation is an important post-translational modification; however, to date, few methylated tubulins have been identified. In the present study, we developed a method for analyzing methylated lysine with the aim of identifying methylated tubulin. This method involves four steps: (1) acid hydrolysis of tubulin into amino acids, (2) selective extraction of methylated lysine using a monolithic-silica disk-packed spin column, (3) fluorescence derivatization of methylated lysine with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and (4) separation of NBD-methylated lysine on a column consisting of C18, cation and anion ligand, and fluorescence detection. Using the newly developed method, the dimethylation of lysine in tubulin was identified. This new method could be applied to searches for other methylated proteins.

中文翻译：

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通过分析甲基化赖氨酸鉴定甲基化微管蛋白

微管蛋白的翻译后修饰（例如乙酰化和去酪氨酸）在微管功能中起重要作用。甲基化是重要的翻译后修饰；然而，迄今为止，几乎没有甲基化的微管蛋白被鉴定出来。在本研究中，我们开发了一种分析甲基化赖氨酸的方法，旨在鉴定甲基化微管蛋白。该方法包括四个步骤：（1）将微管蛋白酸水解为氨基酸；（2）使用硅胶填充的旋转柱选择性萃取甲基化的赖氨酸；（3）用4-fluoro-7对甲基化的赖氨酸进行荧光衍生化-硝基-2,1,3-苯并恶二唑（NBD-F），以及（4）在由C18，阳离子和阴离子配体组成的柱上分离NBD-甲基化赖氨酸，并进行荧光检测。使用新开发的方法，鉴定了微管蛋白中赖氨酸的二甲基化。该新方法可用于搜索其他甲基化蛋白质。