Abstract 2002: Assembling test points for quantitative proteomics to elucidate RAS signaling
Authors: Eric Kuhn, Jeffrey R. Whiteaker, Melissa Martinez, Matthew Holderfield, Jacob Kennedy, Jacob Jaffe, Ping Yan, ChenWei Lin, Gordon Whiteley, Steven A. Carr, Amanda G. Paulovich, John M. Koomen
Journal: Cancer Research (2015)

Abstract RAS mutation is a critical player in cancer development, particularly in lung, colon, and pancreas. However, mutant RAS phenotypes have not produced viable therapeutic options. Therefore, additional reagents and techniques are required to study the functionality of RAS pathway components in model systems and patient specimens. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has been applied to study cancer signaling by quantification of protein expression, mutation status, and post-translational modification in a variety of sample types. LC-MRM with immunoaffinity enrichment (ImmunoMRM) enables multiplexed analysis of target peptides with little background interference. Here, we describe the selection of 100 targets for assay development and provide insight into the utility of the immunoMRM platform as part of the NCI's RAS Initiative. Provided with biologically relevant proteins, several approaches were used for peptide selection. Complementary contributions were obtained from analysis of individual proteins, targeted LC-MRM screening, phosphoproteomics, and global discovery proteomics datasets (both in-house and publically available). For phosphorylation and mutation sites, the ability to quantify those molecular endpoints is directly limited by the primary amino acid sequence produced by trypsin digestion. Peptides selected for assay development quantify biological endpoints indicating the expression levels of wild-type and mutant proteins and signaling of RAS pathway components. Literature reviews show the prospective value of each assay in their specific biological context. Assays are developed for RAS signaling proteins in three categories: upstream proteins, RAS isoforms, and downstream effectors. As an example of upstream proteins, EGFR expression and tyrosine phosphorylation will be quantified, which enables comparison of ImmunoMRM to prior techniques. Extensive characterization is planned for RAS isoforms, particularly to monitor the expression of wild-type and mutant proteins. Measurements focused on downstream proteins include Erk and pErk to examine the activation of that MAPK pathway as well as disparate processes, like epithelial-to-mesenchymal transition (N-Cad/E-Cad). A comprehensive circuit diagram is prepared to illustrate the assembled test points for discussion with the cancer biology community. Development of these immunoMRM assays enables evaluation of multiple RAS signaling events and downstream outcomes in one sample. The emerging paradigm of peptide enrichment for protein biomarker measurement provides a novel platform for the study of critical processes in cancer. Although currently early in development, the implementation of this platform can ultimately demonstrate the utility of ImmunoMRM reagent development and be a first step for understanding RAS biology in human tumors and developing novel treatment options for patients. Citation Format: Eric Kuhn, Jeffrey R. Whiteaker, Melissa Martinez, Matthew Holderfield, Jacob Kennedy, Jacob Jaffe, Ping Yan, ChenWei Lin, Gordon Whiteley, Steven A. Carr, Amanda G. Paulovich, John M. Koomen. Assembling test points for quantitative proteomics to elucidate RAS signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2002. doi:10.1158/1538-7445.AM2015-2002