The AKT/mTOR and RAS/ERK pathways represent mechanisms for cells to regulate survival, proliferation, and motility. These signaling pathways play a central role in tumor progression and drug resistance. Highly accurate monitoring of these pathway proteins has not been achieved, due to poor reproducibility, unreliable quantitation, and lack of standardized methods and reagents. To overcome these challenges, the novel SureQuantTM pathway panels have been applied, which utilize an optimized multiplex immunoprecipitation to a targeted mass spectrometry (mIP-tMS) workflow. mIP-tMS assays can quantitate multiple proteins, PTMs and interacting partners, which creates new possibilities for a broad range of applications, including cancer diagnosis and prognosis, drug development, and precision medicine.
The SureQuantTM AKT pathway (total or phospho), RAS, or TP53 workflow includes a multiplex IP module (antibodies and lysate), MS sample prep, absolute or relative quantitation modules (AQUA Ultimate peptides standards), and standardized data analysis pipeline. Serum-starved, inhibitor-treated (LY294002/NVP-BEZ235/Rapamycin) HCT116, A549, and MCF7 cells were stimulated with hIGF-1. IP-enriched, digested samples were spiked with heavy peptides and analyzed using optimized targeted MS (nanoLC-PRM/MS) and Skyline software. The panels were benchmarked against Western blotting (WB) using three unstimulated, hIGF-1 stimulated or inhibited cell lysates, as well as several tissue lysates.
Previously, we showed the feasibility of optimized mIP-tMS assays to quantitate AKT and RAS pathway proteins across 2 cancer cell lines ± LY294002. The SureQuantTM multiplex pathway panels allowed absolute quantitation of multiple total and phosphorylated targets in low to sub-nanogram concentrations across three unstimulated, hIGF-1 stimulated and inhibited cell lysates as well as tissue/xenograft lysates. Analysis by mass spectrometry allowed for more accurate and informative data with the determination of fmol levels of protein expression and capability to discriminate between isoforms of many proteins that are unable to procure with western blot analysis.
SureQuantTM pathway panels allowed simultaneous absolute quantitation of AKT pathway, RAS proteins and PTMs in a streamlined, standardized workflow.