Abstract 3884: Quantitative analysis of IGF1R/AKT/mTOR pathway using multiplex immunoprecipitation and targeted mass spectrometry

Background: The quantitative measurement of protein expression and modification status of AKT/mTOR pathway proteins is necessary for precise characterization of the disease, monitoring cancer progression and determining treatment response. A major bottleneck in the quantitation of signaling pathway proteins is the lack of rigorously validated methods/reagents and a reliance on semi-quantitative results from current immunoassay technologies (Western blot, ELISA and Luminex). Mass Spectrometry (MS) is increasingly becoming the detection methodology of choice for proteins and their post-translational modifications (PTMs). Immunoprecipitation (IP) is commonly used upstream of MS as an enrichment tool for low-abundant protein targets. In addition to protein identification, IP can be combined with targeted MS to quantitate proteins of interest and identify protein-protein interactions. The objective of this study was to determine the efficacy of multiplex IP to targeted MS technique for measurement of the total and phosphorylated AKT/mTOR pathway targets and to evaluate whether multiplex IP-MS assays are as effective as the current single-plex immunoassay (WB and ELISA) and multiplex Luminex assays. Methods: Serum starved HCT116, MCF7 and A549 cells were stimulated with IGF-1. Multiplex IP to targeted MS assays (mIP-tMS) were developed and validated for absolute quantitation of eleven total and ten phosphorylated AKT/mTOR pathway targets (IGF1R, IR, IRS1, PTEN, AKT, mTOR, GSK3α, GSK3β, TSC2, p70S6K and PRAS40). Validated mIP-tMS assays were benchmarked against currently available WB, ELISA and multiplex Luminex immunoassays across three unstimulated and IGF-1 stimulated cell lysates. Results: In previous work, we showed that an optimized IP-MS workflow for Protein A/G and Streptavidin magnetic beads can increase target protein yield with low non-specific background. In this study, we validated multiple antibodies for eleven total and ten phosphorylated AKT/mTOR pathway targets using the optimized IP-MS workflow. mIP-tMS assays allowed absolute quantitation for all eleven total and ten phosphorylated targets in low to sub nanogram concentrations across three unstimulated and IGF-1 stimulated cell lysates. The benchmarking of mIP-tMS assays showed high correlation for quantitation of total target relative abundance compared to WB, ELISA and Luminex assays. However, for some phosphorylated targets, mIP-tMS assays had low concordance to the other immunoassays possibly due to differences in the specificity of anti-phospho antibodies used for each assay. Conclusion: Overall, the multiplex targeted MS assay can be used for identification and quantification of AKT/mTOR pathway proteins in cancer cell lines or tissue samples. Major advantages of this mIP-tMS assay are high confidence in target identity coupled with simultaneous quantitation of multiple targets and their PTMs. Citation Format: Bhavin Patel, Alex Behling, Leigh Foster, Ryan Bomgarden, Carrie Clothier, Kay Opperman, John Rogers. Quantitative analysis of IGF1R/AKT/mTOR pathway using multiplex immunoprecipitation and targeted mass spectrometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3884.