RESUMO
PURPOSE: Quantitation of ß-tubulin isotype expression in taxane resistant human tumor tissue has been difficult to achieve because of the limited availability of validated antibodies. Here we present a label-free MS method to quantitate relative expression levels of ß-tubulin isotypes. EXPERIMENTAL DESIGN: Using isotype-specific reporter peptides, we determined relative ß-tubulin isotype expression levels in human lung tumor tissue. RESULTS: Four reporter peptides were chosen to quantitate the ßI/ßII, ßIV, ßIII, and ßV tubulin isotypes. These peptides were validated using human cancer cell lines. The label-free method was then used to determine ß-tubulin isotype expression in nine human lung tumor samples, which had been described as high or low ßIII-tubulin expressing using immunohistochemistry. It was found that ßI/ßII (accounting for 18.7-65.7% of total ß-tubulin) and ßIVa/ßIVb (26.3-79.1%) were the most abundant isotypes and that the ßIII (0-8.9%) and ßV (1.0-10.4%) were less abundant in the tissue. We also categorized the samples as high or low ßIII-tubulin expressing. CONCLUSION AND CLINICAL RELEVANCE: With this method we can determine the relative expression levels of ß-tubulin isotypes in human tumor tissue. This method will facilitate studies assessing the use of tubulin isotypes as biomarkers of taxane resistance.
