Abstract : In mammalian brain and ciliary axonemes from ciliates, alpha- and beta-tubulins exhibit an extraordinary heterogeneity due to a combination of multigene family expression and numerous posttranslational modifications (PTMs). The combination of several PTMs located in the C-terminal tail of tubulins plays a major role in this important polymorphism of tubulin: polyglutamylation, polyglycylation, detyrosination, tyrosination, removal of the penultimate glutamate residue, and phosphorylation. In order to document the relationship and functions of these PTMs, we have developed a tubulin C-terminal Peptide Mass Fingerprinting (PMF) method. Using simplified microtubule proteins and tubulin C-terminal peptides purifications, direct matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis can generate a complete picture of all tubulin isotype-specific C-terminal peptides together with their respective PTMs. This chapter will illustrate the capability of this approach to compare tubulin isoform compositions and document the changes in PTMs between samples with different tubulin assembly properties or consecutively to inactivation of modification sites or modification enzymes. Complementary MS-based approaches useful to document the structure of the highly heterogeneous posttranslational polymodifications will also be presented.
