Abstract 1837: Quantitative analysis of AKT/mTOR pathway using immunoprecipitation and targeted mass spectrometry
Authors: Patel Bhavinkumar , Smith Suzanne , Bomgarden Ryan , Opperman Kay , Kaboord Barbara , Rogers John 
Published: August 2015

Abstract
Background: PI3K/AKT/mTOR pathway plays a central role in tumor progression and anti-cancer drug resistance. The quantitative measurement of protein expression and PTM status of AKT/mTOR pathway proteins is necessary for precise characterization of the disease, monitor cancer progression and determine treatment response. A major bottleneck in the quantitation of AKT/mTOR pathway proteins is the lack of rigorously validated methods/reagents and a reliance on semi-quantitative results from Western blotting. Mass Spectrometry (MS) is increasingly becoming the detection methodology of choice for protein abundance and modifications. 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 identify proteins of interest and protein-protein interactions. Here, we used optimized IP-MS to enrich multiple AKT/mTOR pathway proteins simultaneously for targeted selected reaction monitoring (SRM)-based quantitation of protein levels in two human carcinoma cell lines.
Methods: A549 and HCT116 cells were stimulated with EGF or IGF. Several AKT/mTOR pathway targets were enriched by improved IP using Protein A/G and Streptavidin magnetic beads and IP eluates were processed using in-solution digestion for LC-MS analysis. A targeted SRM MS assay was developed for quantitation of AKT/mTOR pathway target peptides (EGFR, AKT2, AKT1, PTEN, PIK3CA, and PIK3R1). Multiple targets were also immunoprecipitated simultaneously and quantitated by targeted SRM assay. Improved IP combined with targeted MS workflow was applied to assess recovery of recombinant proteins in a human plasma matrix.
Results: Immunoprecipitation resulted in overall higher yield of target protein and less non-specific binding compared to unenriched samples. This enabled us to combine multiple target antibodies to simultaneously enrich multiple pathway protein targets. Enrichment of total and phosphorylated forms of EGFR, AKT isoforms, PI3K and PTEN resulted in quantitation of low to sub nanogram levels of targets in two cell lysates by targeted MS (LC-SRM/MS). In addition, IP coupled with targeted MS was used to enrich and quantify as low as 10ng/mL recombinant EGFR, AKT2/AKT1, PTEN and PI3K spiked into a human plasma matrix.
Conclusion: IP combined with targeted MS permits absolute quantitation of AKT/mTOR pathway proteins and PTMs at low to sub nanogram concentrations. This multiplex targeted assay can be used for verification and validation of AKT/mTOR pathway proteins in other cancer cell lines or tissue samples.
Citation Format: Bhavinkumar Patel, Suzanne Smith, Ryan Bomgarden, Kay Opperman, Barbara Kaboord, John Rogers. Quantitative analysis of AKT/mTOR pathway using immunoprecipitation and targeted mass spectrometry. [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 1837. doi:10.1158/1538-7445.AM2015-1837