Characterization of post-translational 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 spectrometry was used to investigate the molecular masses and heterogeneity patterns caused by post-translational modifications in tubulin from porcine brain. Direct analysis of the limited digest with subtilisn shows that the molecular masses of the majority of the carboxyterminal fragments are below 2 kDa, while the truncated tubulin subunits have lost approximately the same mass. The results confirm the cleavage sites previously postulated for this protease. The mass information on the peptides allows the degree of polyglutamylation to be measured directly and shows that molecules with two glutamyl residues in the side chain are the most abundant species. In addition it identifies the degree of tyrosination of alpha tubulin. This one-step monitoring of a complex digest provides information equivalent to that obtainable from the purified components, while the amount of material required is reduced by three orders of magnitude when compared to previous studies. MALDI spectra partially resolve the alpha and beta subunits of the highly homogeneous tubulin from turkey erythrocytes, which lacks polyglutamylation but does not separate alpha and beta subunits from the heterogeneous brain tubulin. Post-translational modifications of the brain tubulin result in shifting peaks to higher molecular masses, in broadening of the peaks, and in loss of resolution.
Rüdiger
A
A
Department of Molecular Structure Research, Gesellschaft für Biotechnologische Forschung, Braunschweig, Federal Republic of Germany.
Rüdiger
M
M
Weber
K
K
Schomburg
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011099:Polyglutamic Acid / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013381:Subtilisins / Q000378:metabolism / Q000494:pharmacology*; D013552:Swine; D014404:Tubulin / Q000097:blood / Q000378:metabolism*; D014422:Turkeys; D014443:Tyrosine / Q000378:metabolism
D005971:Glutamates; D010446:Peptide Fragments; D014404:Tubulin; D011099:Polyglutamic Acid; D014443:Tyrosine; D013381:Subtilisins