Direct Analysis of Tubulin Expression in Cancer Cell Lines by Electrospray Ionization Mass Spectrometry

Pascal Verdier-Pinard, Fang Wang, Berta Burd, Ruth Hogue Angeletti, Susan Band Horwitz, George A. Orr

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

Differential expression of tubulin isotypes, mutations, and/or post-translational modifications in sensitive and Taxol-resistant cell lines suggests the existence of tubulin-based mechanisms of resistance. Since tubulin isotypes are defined by their C-terminal sequence, we previously described a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of tubulin diversity in human cell lines by analysis of their CNBr-released C-terminal peptides [Rao, S., Aberg, F., Nieves, E., Horwitz, S. B., and Orr, G. A. (2001) Biochemistry 40, 2096-103]. We now describe the liquid chromatography/electrospray ionization mass spectrometry analysis of native tubulins in Taxol-stabilized microtubules from parental and Taxol/epothilone-resistant human cancer cell lines. This method allows the direct determination of tubulin isotype composition, including post-translational modifications and mutations occurring throughout the entire protein. Four major isotypes, βI-, βIVb-, Kα1-, and α6-tubulin, were detected in two human carcinoma cell lines, A549 and HeLa. βIII-Tubulin represented a minor species, as did α4-tubulin which was detected for the first time in both cell lines. The three α-tubulins were almost totally tyrosinated, and post-translational modifications were limited to low levels of monoglutamylation of Kα1-, βI-, and βIII-tubulin. βII- and βIVa-tubulins were not detected in either parental or drug-resistant cell lines, in contrast to previous RNA-based studies. Since mutations can occur in a single tubulin allele, the question as to whether the wild-type and mutant transcripts are both translated, and to what levels, is important. Heterozygous expression of Kα1- or βI-tubulin mutants that introduced mass changes as small as 26 Da was readily detected in native tubulins isolated from Taxol- and epothilone-resistant cell lines.

Original languageEnglish (US)
Pages (from-to)12019-12027
Number of pages9
JournalBiochemistry
Volume42
Issue number41
DOIs
StatePublished - Oct 21 2003

ASJC Scopus subject areas

  • Biochemistry

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