A label-free mass spectrometry method for relative quantitation of β-tubulin isotype expression in human tumor tissue

Abstract

Quantitation of β-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 β-tubulin isotypes. Using isotype-specific reporter peptides, we determined relative β-tubulin isotype expression levels in human lung tumor tissue. Four reporter peptides were chosen to quantitate the βI/βII, βIV, βIII, and βV tubulin isotypes. These peptides were validated using human cancer cell lines. The label-free method was then used to determine β-tubulin isotype expression in nine human lung tumor samples, which had been described as high or low βIII-tubulin expressing using immunohistochemistry. It was found that βI/βII (accounting for 18.7-65.7% of total β-tubulin) and βIVa/βIVb (26.3-79.1%) were the most abundant isotypes and that the βIII (0-8.9%) and βV (1.0-10.4%) were less abundant in the tissue. We also categorized the samples as high or low βIII-tubulin expressing. With this method we can determine the relative expression levels of β-tubulin isotypes in human tumor tissue. This method will facilitate studies assessing the use of tubulin isotypes as biomarkers of taxane resistance.