Analysis of Tubulin Isotypes and Mutations from Taxol-Resistant Cells by Combined Isoelectrofocusing and Mass Spectrometry

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Abstract
Six human alpha-tubulin and seven human beta-tubulin isotypes, each of which can undergo posttranslational modifications, have been detected by the reverse transcriptase-polymerase chain reaction. This repertoire of tubulin isotypes plays a role in development and in the building of specialized microtubule-based structures. In cell lines, the relationship between resistance to microtubule-interacting drugs and altered tubulin isotype expression profiles is often established by quantitation of cDNA and/or Western blot analysis. Tubulin mutations in major isotypes are detected by sequencing cDNA, but more analysis of expression of tubulin mutations at the protein level, to assess their role in drug resistance, is needed. We utilized a Taxol-based purification and high-resolution isoelectrofocusing combined with a mass spectrometry-based analysis of tubulin. This approach has allowed the separation and relative quantitation of tubulin isotypes having a difference in isoelectric point values of 0.01, without the need for two-dimensional gel electrophoresis. The specificity of tubulin isotype antibodies also has been established. In cell lines resistant to microtubule-stabilizing drugs that express heterozygous tubulin mutations, the relative amount of mutant tubulin expression has been determined. In these cell lines, the absence of betaII- and betaIVa-tubulin has been demonstrated, and an increased level of expression of betaIII-tubulin in resistant cells has been confirmed, indicating that this tubulin isotype is a unique marker of resistance.