Bhavin Patel1, Penny Jensen1, Abid Haseeb1, Leigh Foster1, Gregory Potts2, Kay Opperman1, Rosa I Viner3, Andreas Huhmer3, John C Rogers1
Introduction: The objective of this study was to determine the efficacy of multiplex IP to targeted MS (mIP-tMS) technique for measurement of the total and phosphorylated AKT/mTOR & RAS/ERK pathway targets and to evaluate whether mIP-tMS assays are as effective as the current Western Blot (WB) techniques.
Methods: Serum starved and LY294002 (PI3K inhibitor) treated HCT116 and A549 cells were stimulated with hIGF-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 protein targets in these pathways and benchmarked with Western blot using unstimulated, hIGF-1 stimulated and LY294002 treated samples for two cell lysates.
Preliminary Data: Previously, we demonstrated that an optimized IP-MS workflow for Protein A/G and Streptavidin magnetic beads increases target protein abundance with low nonspecific background. In this study, we validated antibodies to several AKT/mTOR and RAS/ERK pathway protein targets using an optimized IP-MS workflow. mIP-tMS assays allowed for absolute quantitation of multiple total and phosphorylated protein targets from both pathways in low to sub-nanogram concentrations across unstimulated, hIGF-1 stimulated and LY294002 treated samples for two cell lysates. The benchmarking of mIP-tMS assays indicated target dependant correlation for quantitation of total and phosphorylated protein targets relative to Western blot. Targets with low correlation between techniques may be caused by differences in the specificity of antibodies used for each assay technique.
1 Thermo Fisher Scientific, Rockford, IL
2 Abbvie, Abbott Park, IL,
3 Thermo Fisher Scientific, San Jose, CA
United States
