CHARACTERIZATION OF POSTTRANSLATIONAL MODIFICATIONS OF BRAIN TUBULIN BY MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS-SPECTROMETRY - DIRECT ONE-STEP ANALYSIS OF A LIMITED SUBTILISIN DIGEST/

Matrix-assisted ultraviolet laser desorption/ionization (MALDI) mass s pectrometry was used to investigate the molecular masses and heterogen eity patterns caused by post-translational modifications in tubulin fr om porcine brain. Direct analysis of the limited digest with subtilisi n shows that the molecular masses of the majority of the carboxytermin al fragments are below 2 kDa, while the truncated tubulin subunits hav e lost approximately the same mass, The results confirm the cleavage s ites previously postulated for this protease. The mass information on the peptides allows the degree of polyglutamylation to be measured dir ectly and shows that molecules with two glutamyl residues in the side chain are the most abundant species. In addition it identifies the deg ree of tyrosination of alpha tubulin. This onestep monitoring of a com plex digest provides information equivalent to that obtainable from th e purified components, while the amount of material required is reduce d by three orders of magnitude when compared to previous studies. MALD I spectra partially resolve the alpha and beta subunits of the highly homogeneous tubulin from turkey erythrocytes, which lacks polyglutamyl ation but does not separate alpha and beta subunits from the heterogen eous brain tubulin. Post-translational modifications of the brain tubu lin result in shifting peaks to higher molecular masses, in broadening of the peaks, and in loss of resolution. (C) 1995 Academic Press, Inc .