Identification by mass spectrometry of a new alpha-tubulin isotype expressed in human breast and lung carcinoma cell lines

The extensive C-terminal molecular heterogeneity of alpha- and beta -tubuli n is a consequence of multiple isotypes, the products of distinct genes, th at undergo several posttranslational modifications. These include polygluta mylation and polyglycylation of both subunits, reversible tyrosination and removal of the penultimate glutamate from alpha -tubulin, and phosphorylati on of the beta III isotype. A mass spectrometry-based method has been devel oped for the analysis of the C-terminal diversity of tubulin from human cel l lines. Total cell extracts are resolved by SDS-PAGE and transferred to ni trocellulose, and the region of the blot corresponding to tubulin (similar to 50 kDa) was excised and digested with CNBr to release the highly diverge nt C-terminal tubulin fragments. The masses of the human alpha- and beta -t ubulin CNBr-derived C-terminal peptides are all in the 1500-4000 Da mass ra nge and can be analyzed directly by MALDI-TOF mass spectrometry in the nega tive ion mode without significant interference from other released peptides . In this study, the tubulin isotype diversity in MDA-MB-231, a human breas t carcinoma cell line, and A549, a human non-small lung cancer cell line, i s reported. The major tubulin isotypes present in both cell lines are k-alp ha1 and beta1. Importantly, we report a previously unknown alpha isotype pr esent at significant levels in both cell lines. Moreover, the degree of pos ttranslational modifications to all isotypes was limited. Glu-tubulin, in w hich the C-terminal tyrosine of alpha -tubulin is removed, was not detected . In contrast to mammalian neuronal tubulin which exhibits extensive polygl utamylation, only low-level monoglutamylation of the k-alpha1 and beta1 iso types was observed in these two human cell lines.