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

• Published in: Biochemistry (Easton) 2003-10, Vol.42 (41), p.12019-12027
• Main Authors: Verdier-Pinard, Pascal, Wang, Fang, Burd, Berta, Angeletti, Ruth Hogue, Horwitz, Susan Band, Orr, George A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Cell Line, Tumor; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; Epothilones - pharmacology; HeLa Cells; Humans; Molecular Sequence Data; Molecular Weight; Mutation; Neoplasm Proteins - biosynthesis; Neoplasm Proteins - genetics; Neoplasm Proteins - isolation & purification; Paclitaxel - pharmacology; Peptide Fragments - genetics; Peptide Fragments - isolation & purification; Peptide Mapping; Protein Isoforms - biosynthesis; Protein Isoforms - genetics; Protein Isoforms - isolation & purification; Spectrometry, Mass, Electrospray Ionization - methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin; Tubulin - biosynthesis; Tubulin - genetics; Tubulin - isolation & purification
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0350147

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.