on 10-30-201806:49 AM - edited on 10-15-202106:27 AM by Closed Account
Penny Jensen1; Bhavin Patel1; Leigh Foster1; Renuka Sabnis1; Aaron Gajadhar2; Jonathan R. Krieger3; Jiefei Tong3; Michael F. Moran3,4; Ming S. Tsao5, Rosa Viner2; Andreas Huhmer2; Kay Opperman1; John Rogers1;
Purpose: To develop a robust method for quantitation of AKT pathway proteins and benchmark against western blotting.
Methods: The SureQuantTM AKT pathway (total or phospho) panels include a multiplex immunoprecipitation (IP) and mass spectrometry (MS) sample prep module (antibodies, lysate, buffers), absolute or relative quantitation modules (AQUA Ultimate peptides standards), and instrument method /Skyline software templates. Serum starved, inhibitor-treated (LY294002/Rapamycin/ NVP-BEZ235) A549, MCF7, and HCT116 cells were stimulated with hIGF-1 and prepared for MS analysis using the SureQuantTM AKT total and phospho multiplex pathway modules in order to determine the absolute concentration of pathway peptides using targeted MS analysis. The panels were benchmarked against Western blotting (WB) using cell lysates as well as tissue/xenograft lysates.
Results: The SureQuantTM multiplex pathway modules achieved absolute quantitation of multiple total and phosphorylated targets across unstimulated, hIGF-1 stimulated and inhibited + hIGF-1 stimulated cell lysates as well as tissue/xenograft lysates. Benchmarking of SureQuantTM AKT Pathway Targeted MS assays relative to WB showed overall good correlation between orthogonal techniques. Any discrepancies between two techniques may be result from differences in the antibodies used for each assay.
1 Thermo Fisher Scientific, Rockford, IL
2 Thermo Fisher Scientific, San Jose, CA
3 The Hospital for Sick Children, Toronto, Canada
4 Department of Molecular Genetics, University of Toronto, Canada
6 Princess Margaret Cancer Centre, Toronto, Canada