Unlocking the activities of polyglutamylation enzymes in brain tubulin with top-down mass spectrometry

Microtubules, essential components of the cytoskeleton, are formed from a− and b−tubulin heterodimers which are organized in a cylindrical fashion, like a barrel. The C-terminal tubulin tails remain exposed on the outside of the MT, where they can be highly modified by post-translational modifications (PTMs), including glutamylation, which modulates the interaction of tubulin with other proteins. The addition and extension of polyglutamate chains are regulated by tubulin tyrosine ligase-like (TTLL) enzymes. In mice, TTLL7 is primarily responsible for the addition of the first glutamate residue, and TTLL1 acts as an extender, adding more glutamates to form a polyglutamate chain. By analyzing tubulin from wild-type, TTLL1-KO, TTLL7-KO, and double-KO mouse brains using top-down mass spectrometry (TD-MS), we demonstrate the distinct and non-redundant enzymatic specificities of TTLL1 and TTLL7: TTLL1 exclusively modifies a−tubulin, while TTLL7 targets b−tubulin. Deletion of each enzyme leads to the selective loss of polyglutamylation in the corresponding tubulin, with no compensation by other TTLLs. In total, we identified 47 proteoforms, with up to 5 glutamylations, verified by targeted MS/MS fragmentation. Despite challenges posed by the high sequence homology and acidic C-terminal tails of tubulins, our automated TD-MS pipeline enables rapid proteoform identification in complex samples. This approach provides, for the first time, a comprehensive snapshot of the glutamylation status of intact tubulins, paving the way for quantitative studies of the tubulin code. Our method, adaptable to other enzymes, reveals critical enzymatic roles in microtubule regulation and highlights the utility of TD MS for studying challenging protein targets.