Abstract 1841: Enrichment of IGF1R-AKT-mTOR pathway proteins using immunoprecipitation and proteomic analysis by mass spectrometry

Abstract

Abstract Background: A major bottleneck in the proteomic characterization of signaling pathway targets is the lack of methods/reagents to quantify medium to low levels of proteins of interest in human samples. Immunoprecipitation followed by mass spectrometry (IP-MS) enables the simultaneous analysis of protein expression, protein-protein interactions and post-translational modifications (PTMs). Immunoprecipitation provides both enrichment and increased sensitivity while the MS provides specific identification, high selectivity and multiplex possibility. Dysregulation of IGF1R -PI3K-AKT-mTOR signaling is a pivotal driver for many cancers making this pathway an attractive therapeutic target for cancer therapy. The quantitative evaluation of protein expression and PTM status of these signal transduction proteins will aid in the precise characterization of the disease at the diagnostic or prognostic level and to monitor its progression and treatment response. Methods: To investigate the IGF1R-PI3K-AKT-mTOR signaling pathway, we used stable isotopic amino acids in cell culture (SILAC) to differentially label proteins for quantification in insulin growth factor (IGF) stimulated versus unstimulated A549 and HCT116 cells. Targets were immunoprecipitated from the cell lysates, captured on streptavidin or Protein A/G magnetic beads, and eluted from the beads for direct in-solution tryptic digestion. Quantitative changes in total and phosphorylated forms of AKT-mTOR pathway targets were evaluated by liquid chromatography-mass spectrometry (LC-MS) on a Thermo Scientific Orbitrap Fusion platform. Results: The optimized IP-MS protocol resulted in the identification of low copy number proteins that could not be detected by LC-MS analysis without enrichment. Some of the low abundance targets observed after enrichment included PI3K, PTEN, and AKT isoforms present at low to sub nanogram levels. Interacting proteins including the catalytic and regulatory subunits of PI3K were also identified indicating that the workflow supports co-immunoprecipitation of protein complexes. Enrichment of several IGF1R-PI3K-AKT-mTOR pathway targets (IGF1R, PI3KCA, PIK3R2, AKT, and PTEN) from two cell lysates facilitated the relative quantification of total and phosphorylated forms of these proteins by LC-MS analysis. Conclusion: SILAC combined with the improved IP-MS method permits relative quantification of specific and functional protein-protein interactions and PTMs in the IGF1R-PI3K-AKT-mTOR pathway, which can ultimately lead to better cancer characterization and monitoring. Citation Format: Suzanne Smith, Bhavin Patel, Ryan Bomgarden, Kay Opperman, John Rogers, Barbara Kaboord. Enrichment of IGF1R-AKT-mTOR pathway proteins using immunoprecipitation and proteomic analysis by 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 1841. doi:10.1158/1538-7445.AM2015-1841