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A label-free mass spectrometry method for relative quantitation of β-tubulin isotype expression in human tumor tissue
Leah M Miller1, Chia-Ping Huang Yang, Hui Xiao
1Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Purpose:
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.
Experimental Design:
Using isotype-specific reporter peptides, we determined relative β-tubulin isotype expression levels in human lung tumor tissue.
Results:
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.
Conclusion And Clinical Relevance:
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.
Insights
A new label-free mass spectrometry method accurately quantifies beta-tubulin isotype expression in human lung tumors. This technique aids in identifying biomarkers for taxane resistance, overcoming limitations of antibody-based methods.
Area of Science:
- Biochemistry
- Proteomics
- Cancer Biology
Background:
- Quantifying beta-tubulin isotype expression in taxane-resistant human tumors is challenging due to a lack of validated antibodies.
- Beta-tubulin isotypes play crucial roles in microtubule dynamics and are implicated in cancer drug resistance.
Purpose of the Study:
- To develop and validate a label-free mass spectrometry (MS) method for quantifying relative beta-tubulin isotype expression levels.
- To assess the utility of this method in human lung tumor tissue.
Main Methods:
- Development of isotype-specific reporter peptides for beta-tubulin isotypes (βI/βII, βIV, βIII, βV).
- Validation of reporter peptides using human cancer cell lines.
- Application of a label-free MS approach to analyze nine human lung tumor samples.
Main Results:
- The label-free MS method successfully quantified relative beta-tubulin isotype expression.
- Beta-tubulin isotypes βI/βII (18.7-65.7%) and βIVa/βIVb (26.3-79.1%) were the most abundant in lung tumors.
- Beta-tubulin isotypes βIII (0-8.9%) and βV (1.0-10.4%) were less abundant, with samples categorized as high or low βIII expressers.
Conclusions:
- The developed label-free MS method enables accurate determination of relative beta-tubulin isotype expression in human tumor tissue.
- This method provides a valuable tool for future research into beta-tubulin isotypes as potential biomarkers for taxane resistance.
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