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.
Verdier-Pinard
Pascal
P
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Wang
Fang
F
Burd
Berta
B
Angeletti
Ruth Hogue
RH
Horwitz
Susan Band
SB
Orr
George A
GA
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D019008:Drug Resistance, Neoplasm; D034261:Epothilones / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D017239:Paclitaxel / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D010449:Peptide Mapping; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin; D014404:Tubulin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D034261:Epothilones; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D014404:Tubulin; D014357:Trypsin; D017239:Paclitaxel