Abstract 1615: Enrichment of EGFR/PI3K/AKT/PTEN proteins using immunoprecipitation and analysis with mass spectrometry-based proteomics
Authors: Patel Bhavinkumar , Meier Scott , Opperman Kay , Haney Paul , Kaboord Barbara , Rogers John 
Published: May 2015

Abstract
Background: The dynamic range of protein concentrations spans more than seven orders of magnitude in human cell lines and eleven orders of magnitude in human plasma/serum and tissue. Currently, a major bottleneck in the verification of candidate cancer biomarkers 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) allows for the simultaneous analysis of protein expression, protein-protein interactions and post-translational modifications (PTMs) of protein biomarkers. The immunoaffinity aspect provides both enrichment and increased sensitivity while the mass spectrometry provides high specificity, high selectivity and multiplex possibility. Dysregulated EGFR-PI3K-AKT signaling is considered pivotal for many cancers, and this pathway is a potential candidate for therapeutic targeting. The quantitative evaluation of protein expression and PTM status of these and other signal transduction protein targets in cell lines, tissue, and plasma will enable precise characterization of the disease at the diagnostic or prognostic level and to monitor its progression and treatment response.
Methods: Here we evaluated immunoprecipitation with directly coupled antibody or biotinylated antibody with immobilized streptavidin resin for MS applications. We evaluated several beads/resins to precipitate EGFR from A431 cell lysate with subsequent detection and quantitation by mass spectrometry. EGFR, PI3K, AKT isoforms and PTEN were immunoprecipitated from two cell lysates using the optimized IP to MS workflow. A multiplex targeted assay (LC-SRM/MS) was developed and implemented to measure limit of quantitation (LLOQ) of the EGFR, PI3K, AKT2, AKT1, and PTEN tryptic peptides in two cell lysates and plasma.
Results: Immunoprecipitation using magnetic beads resulted in overall higher yield of target protein and less non-specific binding for MS applications. Enrichment of EGFR, AKT isoforms, and PTEN in two cell lysates permitted detection by discovery MS and quantitation by targeted MS. Immunoprecipitation of EGFR and AKT2 resulted in simultaneous analysis of multiple phosphorylation sites. EGFR, AKT1, AKT2 and PTEN were quantitated in low nanogram range by LC-SRM/MS in two cell lysates. Enrichment of as low as 7ng/mL recombinant EGFR in human plasma matrix allowed absolute quantitation by targeted MS.
Conclusion: The application of IP to the analysis of EGFR, PI3K, AKT and PTEN protein targets permits detection and quantitation of protein and PTMs at sub to low ng/mL concentrations with mass spectrometry approaches.
Citation Format: Bhavinkumar Patel, Scott Meier, Kay Opperman, Paul Haney, Barbara Kaboord, John Rogers. Enrichment of EGFR/PI3K/AKT/PTEN proteins using immunoprecipitation and analysis with mass spectrometry-based proteomics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1615. doi:10.1158/1538-7445.AM2014-1615