Quantitative Analysis of AKT/mTOR Pathway using Multiplex-Immunoprecipitation and Targeted Mass Spectrometry

摘要

Introduction: To determine the efficacy of multiplex IP to targeted MS (mIP-tMS) technique for measurement of the total and phosphorylated AKT-mTOR pathway targets and to evaluate whether mIP-tMS assays are as effective as the current singleplex immunoassays (Western Blot (WB) and ELISA) as well as multiplex Luminex assays. Methods: Serum starved HCT116, MCF7 and A549 cells were stimulated with IGF-1. mIP-tMS assays were developed and validated for absolute quantitation of 12 total and 11 phosphorylated AKT/mTOR pathway targets. Validated mIP-tMS assays were benchmarked against currently available WB, ELISA and multiplex Luminex immunoassays across 3 unstimulated and IGF-1 stimulated cell lysates. Preliminary Data: In previous work, we showed that an optimized IP-MS workflow for Protein A/G and Streptavidin magnetic beads can increase target protein yield and lowers non-specific background. We validated multiple antibodies for 12 total and 11 phosphorylated AKT/mTOR pathway targets using the optimized IP-MS workflow. mIP-tMS assays allowed absolute quantitation for all 12 total and 11 phosphorylated targets in low to sub-nanogram concentrations across all cell lines. The benchmarking of mIP-tMS assays showed moderate correlation for quantitation for 12 total and 11 phosphorylated target relative abundance compared to WB, ELISA and Luminex assays. Novel Aspects: 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 MS assay are high confidence in target identity coupled with simultaneous quantitation of multiple targets and their PTMs.