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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Direct analysis of tubulin expression in cancer cell lines by electrospray ionization mass spectrometry.

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, betaI-, betaIVb-, Kalpha1-, and alpha6-tubulin, were detected in two human carcinoma cell lines, A549 and HeLa. betaIII-Tubulin represented a minor species, as did alpha4-tubulin which was detected for the first time in both cell lines. The three alpha-tubulins were almost totally tyrosinated, and post-translational modifications were limited to low levels of monoglutamylation of Kalpha1-, betaI-, and betaIII-tubulin. betaII- and betaIVa-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 Kalpha1- or betaI-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.[1]


  1. Direct analysis of tubulin expression in cancer cell lines by electrospray ionization mass spectrometry. Verdier-Pinard, P., Wang, F., Burd, B., Angeletti, R.H., Horwitz, S.B., Orr, G.A. Biochemistry (2003) [Pubmed]
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