Mass spectrometry reveals novel features of tubulin polyglutamylation in the flagellum of Trypanosoma brucei

Tubulin polyglutamylation is a prominent feature of eukaryotic cilia and flagella. In the protist parasite Trypanosoma brucei , nine different tubulin tyrosine ligase-like (TTLL) enzymes are potentially involved in tubulin glutamylation. Given this enzymatic diversity, it is important to qualitatively and quantitatively characterize tubulin polyglutamylation, in order to understand the trypanosome tubulin code and pave the way for functional assignments of different TTLLs. Robust mass spectrometry (MS) based proteomics approaches for analysis of posttranslational modifications have been developed in recent years, but none to study polyglutamylation. We therefore optimized a nanoLC-MS/MS pipeline, from sample preparation to data analysis, using synthetic polyglutamylated peptides for quantification. Applied to the flagellum of T. brucei, our approach enabled the detection and quantification of C-terminal tubulin peptides with up to eleven supplementary glutamates on α-, and five on β-tubulin. In addition to the known E445 on α- and E435 on β-, a novel glutamylation site of β-tubulin was discovered at residue E438. Furthermore, our data revealed an increase in enzymatic detyrosination with increasing length of the glutamate chains, especially for α-tubulin. This indicates crosstalk between the modifications and different rates of detyrosination of the two tubulin types. Our results complement the existing knowledge of the tubulin code in T. brucei and introduce an efficient analytical pipeline for the characterization of polyglutamylated proteins.