Penny Jensen1; Bhavin Patel1; Leigh Foster1; Aaron Gajadhar2; Sebastien Gallien3; Jonathan R. Krieger4; Jiefei Tong5; Michael F. Moran4,5,6; Rosa Viner2; Andreas Huhmer2; Kay Opperman1; John Rogers1
ASMS 2019 Poster
Purpose: The AKT/mTOR pathway plays a central role in tumor progression and drug resistance. Quantitative measurement of alterations in the expression of pathway proteins and post-translational modifications (PTM) is necessary for understanding cancer biology. 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 targeted mass spectrometry (mIP-tMS) workflow. SureQuant assays can quantitate multiple proteins, PTMs and interacting partners, which creates new possibilities for a broad range of applications, including cancer, drug development, and research into precision medicine.
Methods: The SureQuant total and phospho pathway panels contain two modules: 1) The IP-MS Sample Prep Module includes reagents necessary to immunoenrich AKT pathway, RAS, or TP53 proteins, and perform MS sample preparation in one day 2) The Absolute or Relative Quantitation Modules include a Pierce™ LC-MS/MS System Suitability Standard, AQUA Ultimate Heavy and/or AQUA Ultimate Light Peptides, and verified MS instrument and data analysis methods. Serum-starved, inhibitor-treated (LY294002/NVP-BEZ235/Rapamycin) HCT116, A549, and MCF7 cells were stimulated with hIGF-1. SureQuant AKT pathway panels (total and phospho) were used to determine the absolute concentration of target peptides using targeted MS analysis. The panels were benchmarked against Western blotting using three unstimulated, hIGF-1 stimulated or inhibited cell lysates, as well as several tissue/xenograft lysates.
Results: Previously, we verified antibodies and target peptides to AKT and RAS pathways using an optimized mIP-tMS workflow. From the standard curve, all target peptides were monitored with <20% CV, 3 orders of magnitude dynamic range, linearity (R2) >0.97, and accuracy of 80-120% in a complex matrix. Using the SureQuantTM pathway panels, absolute quantitation of 37 target peptides in unknown samples was achieved with <20% CV across multiple cancer cell lines. The SureQuant pathway analysis workflow allowed absolute quantitation of target peptides from positive control lysate with <15% individual operator %CV and <20% combined %CV using PRM analysis. Kit performance was evaluated through analysis of abundance levels between three different cancer cell lines, A549, HCT116, and MCF7, using the SureQuant AKT Total and Phospho assay showed preferences for certain inhibitors in specific cell lines treated with hIGF-1. The PI3K inhibitor LY294002 functioned the best in HCT116 cells whereas the dual PI3K/Rapamycin inhibitor NVP-BEZ235 worked predominantly in A549 cells. 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. Absolute quantitation of 12 phosphorylated AKT pathway targets was obtained from five patient derived lung tumor xenograft samples. Additionally, all 12 total and 12 phospho AKT pathway targets were quantitated from three different tissue lysates, Lung, Large Intestine, and Breast tumor.
1Thermo Fisher Scientific, Rockford, IL;Â 2Thermo Fisher Scientific, San Jose, CA; 3Thermo Fisher Scientific, PMSC, Cambridge, MA; 4SPARC Biocentre, The Hospital for Sick Children, Toronto, Canada; 5Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada; 6Department of Molecular Genetics, University of Toronto, Canada