Mass Spectrometry Reveals Novel Features of Tubulin Polyglutamylation in the Flagellum of Trypanosoma brucei

Tubulin polyglutamylation is a key feature of eukaryotic cilia and flagella that is essential for their function. The diversity of enzymes catalyzing polyglutamylation with different specificities inspired the hypothesis of the tubulin code. In the protist parasite Trypanosoma brucei, nine different glutamylase enzymes are potentially involved in tubulin glutamylation. To decipher the trypanosome tubulin code generated by this diversity, we aimed at determining tubulin glutamylation patterns by robust mass spectrometry (MS)-based proteomics. MS approaches exist for many post-translational modifications but none for the chemically complex polyglutamylation. We therefore optimized a nanoLC-MS/MS pipeline from sample preparation to data analysis using synthetic peptides for quantification. Our approach enabled the quantification of C-terminal tubulin peptides with up to 11 supplementary glutamates on α-, and five on β-tubulin from the flagellum of T. brucei. In addition to the known E445 on α- and E435 on β-, a novel glutamylation site of β-tubulin was discovered at E438. Furthermore, our data revealed an increase in enzymatic detyrosination with increasing length of the glutamate chains, especially for α-tubulin. This indicates cross-talk between the modifications and different detyrosination rates of the two tubulin types. Our efficient analytical pipeline advances understanding of the tubulin code in T. brucei.