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Updated: Aug 30, 2026
Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors
Published on: February 27, 2015
Direct analysis of tubulin expression in cancer cell lines by electrospray ionization mass spectrometry
Pascal Verdier-Pinard1, Fang Wang, Berta Burd
1Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
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, 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.
Insights
Tubulin mutations and modifications contribute to cancer drug resistance. Liquid chromatography/electrospray ionization mass spectrometry directly analyzes tubulin in resistant cells, detecting mutations and post-translational modifications.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Tubulin isotype expression, mutations, and modifications are implicated in Taxol resistance.
- Previous C-terminal peptide analysis using mass spectrometry provided insights into tubulin diversity.
- Direct analysis of native tubulins offers a more comprehensive understanding of resistance mechanisms.
Purpose of the Study:
- To directly analyze native tubulin composition, including post-translational modifications and mutations, in drug-sensitive and drug-resistant human cancer cell lines.
- To compare tubulin profiles between parental and resistant cell lines using liquid chromatography/electrospray ionization mass spectrometry.
- To investigate the translation of mutant tubulin alleles in resistant cell lines.
Main Methods:
- Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) was employed to analyze native tubulins.
- Taxol-stabilized microtubules were isolated from parental and resistant human cancer cell lines (A549 and HeLa).
- Analysis focused on the direct determination of tubulin isotype composition, post-translational modifications, and mutations.
Main Results:
- Four major tubulin isotypes (betaI-, betaIVb-, Kalpha1-, and alpha6-tubulin) were identified in A549 and HeLa cells.
- betaIII- and alpha4-tubulin were detected as minor species; alpha4-tubulin was identified for the first time.
- Alpha-tubulins were highly tyrosinated, with low levels of monoglutamylation observed. betaII- and betaIVa-tubulins were notably absent.
- Heterozygous expression of Kalpha1- or betaI-tubulin mutants, even with small mass changes, was detected in resistant cell lines.
Conclusions:
- Direct mass spectrometry analysis of native tubulins provides a comprehensive view of tubulin composition and modifications.
- The study identified specific tubulin isotypes and modifications in human carcinoma cell lines.
- Detection of heterozygous tubulin mutants confirms their translation and potential role in drug resistance.
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