Enzymatic modification of tubulins: Tubulin was treated with carboxypeptidase and with tubulin tyrosine ligase as described previously . Briefly, tubulin in PME buffer (0.1 M PIPES, 1 mM MgSO4, 2 mM EGTA, pH 6.9) was treated with CPA (final concentrations: 0.1 g/L CPA, 3 g/L tubulin) at 37 ºC for 30 min. To quench the reaction, DTT (final concentration: 20 mM) was added and the solution was incubated on ice for 20 min. The cleaved tyrosine residues were removed, and the protein was equilibrated in TTL buffer (25 mM MES, 150 mM KCl, 27 µM MgCl2, 2.5 mM ATP, 1 mM DTT, 1.5% glycerol, pH 6.8) by rapid gel filtration chromatography using Sephadex G50 resin in TTL buffer. Tubulin re-tyrosination was performed by incubating the detyrosinated tubulin with excess tyrosine and recombinant GST-tubulin tyrosine ligase (final concentrations: 0.2 g/L GST-TTL, 1 mM tyrosine) at 37 ºC for 30 min prior to SDS-PAGE or cyanogen bromide (CNBr) cleavage and preparation for mass spectrometry. VASH1/SVBP digestion of tubulin was performed as described as described . Protein was then processed for mass spectrometry as described above.