Identification of methylated tubulin through analysis of methylated lysine.

Suzuki, Rika; Funatsu, Takashi; Tsunoda, Makoto

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.

MICROTUBULES; LYSINE; METHYLATION; ACETYLATION; AMINO acids

Analytical & Bioanalytical Chemistry, 2018, Vol 410, Issue 17, p4189

1618-2642

Academic Journal

10.1007/s00216-018-1068-9