Abstract 217: Quantitative analysis of two cancer signaling pathways using multiplex immunoprecipitation and targeted mass spectrometry

Abstract

Abstract Background: The AKT/mTOR and RAS/ERK pathways represent key mechanisms for cells to regulate cell survival, proliferation, and motility. In addition to their independent signaling cascades, which provide offsetting mechanisms, these two pathways extensively engage in cross-talk in order to both positively and negatively regulate each other. The quantitation of protein expression and modifications of pathway targets are critical for characterization of disease, monitoring cancer progression and determining treatment response. Some major bottlenecks in the accurate quantitation of these proteins are the lack of rigorously validated reagents and a reliance on semi-quantitative results from available immunoassays. Mass spectrometry (MS) is increasingly becoming the detection method of choice for proteins and their post-translational modifications (PTMs). Immunoprecipitation coupled with MS (IP-MS) enables assessment of antibody specificity and identification of low-abundant targets. Multiplexed IP coupled with targeted MS (mIP-tMS) can quantitate multiple proteins of interest, PTMs and interacting partners in a single MS run. The objective of this study was to determine the efficacy of mIP-tMS for quantitation of the AKT/mTOR and RAS/ERK pathway proteins and to compare these results versus Western blotting (WB). Methods: Serum starved and LY294002 treated HCT116 and A549 cells were stimulated with IGF-1. Antibodies to targets in the AKT/mTOR and RAS/ERK pathways were selected for verification of antibody specificity by IP-MS. mIP-tMS assays were developed and validated for absolute quantitation of targets in these pathways and benchmarked with WB across two unstimulated, IGF-1 stimulated and LY294002 treated cell lysates as well as several tissue lysates. Results: Previously, we showed that an optimized IP-MS workflow for Protein A/G and Streptavidin magnetic beads increases target protein yield with low non-specific background. In this study, we validated antibodies to several AKT/mTOR and RAS/ERK pathway targets using the optimized IP-MS workflow. mIP-tMS assays allowed absolute quantitation for multiple total and phosphorylated targets from both pathways in low to sub-nanogram concentrations across two unstimulated, IGF-1 stimulated and LY294002 treated cell lysates. The benchmarking of mIP-tMS assays showed low correlation for quantitation of total and phosphorylated targets relative to WB. This lower correlation may be due to differences in the specificity of antibodies used for each assay technique. Conclusion: Overall, the mIP-tMS assays can be used for effective identification and quantitation of AKT/mTOR and RAS/ERK pathway proteins in multiple cancer cell lines and tissue samples. Major advantages of this assay are high confidence in target identity coupled with simultaneous absolute quantitation of multiple targets and their PTMs from cancer signaling pathways. Note: This abstract was not presented at the meeting. Citation Format: Bhavin Patel, Leigh Foster, Gregory Potts, Abid Haseeb, Alex Behling, John Rogers. Quantitative analysis of two cancer signaling pathways using multiplex immunoprecipitation and targeted mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 217. doi:10.1158/1538-7445.AM2017-217