Abstract 2491: Quantification of biomarker expression, phosphorylation, and mutation in cancer using TMT labeling prior to liquid chromatography-multiple reaction monitoring mass spectrometry
Authors: Chen Yi , Britton David J. , Paraiso Kim , Fedorenko Inna , Wood Elizabeth R. , Magliocco Anthony , Sondak Vernon K. , Smalley Keiran , Pike Ian , Koomen John M. 
Published: May 2015

Abstract
Introduction: Quantitative mass spectrometry assays using the Tandem Mass Tag (TMT) labeling strategy have been developed for measuring biologically relevant phosphorylation and mutation sites, which are not easily amenable to quantification in standard proteomics workflows. Quantification of the c-terminal peptide from HER2, which can be phosphorylated at Tyrosine 1248, reports on potential binding of Shc and activation of Ras signaling. In melanoma, N-Ras mutations at Q61 can occur de novo or develop as a resistance mechanism to B-Raf inhibition. However, tryptic peptides containing these specific sites have poor or no signal for liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) quantification. Different proteolysis strategies and TMT labeling enhanced the signal intensity of those specific peptides and enabled quantification of these candidate biomarkers. The quantification of target peptides with specific phosphorylation and mutation sites will help to understand cancer biology and may predict drug response to support individualized selection of therapy.
Experimental Procedures: Model systems included breast cancer (MCF7, BT474) and melanoma (N-Ras WT M257, N-Ras Q61K M245, N-Ras Q61R 2032, N-Ras Q61L M318) cell lines inter al. Peptide-based quantitative assays were developed for HER2 expression and phosphorylation as well as N-Ras expression and mutation using TMT labeling prior to LC-MRM analysis. Endogenous peptides including wild type N-Ras and all known Q61 mutations have been synthesized and tested for TMT labeling efficiency with LC-MRM. Then, proteins were fractionated by SDS-PAGE from whole cell lysates. Different proteolytic strategies including chymotrypsin or Glu-C digestion were optimized for biomarker quantification. The assay characteristics were evaluated using serial dilutions of cell lysate before analysis of clinical specimens.
Data Summary: LC-MRM assays for both unmodified and phosphorylated HER2 as well as wild type and mutant N-Ras have been developed and characterized using cell line models. TMT labeling efficiency has been evaluated under different conditions to accommodate different digestion buffers. Assays have been implemented to study these biomarkers.
Conclusions: Proteomic quantification of cancer biomarkers defined by known biology, including expression, phosphorylation, and mutation status, has the potential to improve the selection of targeted therapeutics and prediction of drug response.
Citation Format: Yi Chen, David J. Britton, Kim Paraiso, Inna Fedorenko, Elizabeth R. Wood, Anthony Magliocco, Vernon K. Sondak, Keiran Smalley, Ian Pike, John M. Koomen. Quantification of biomarker expression, phosphorylation, and mutation in cancer using TMT labeling prior to liquid chromatography-multiple reaction monitoring mass spectrometry. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2491. doi:10.1158/1538-7445.AM2014-2491