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

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

SUBMITTER: Miller LM 

PROVIDER: S-EPMC3484181 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature