on 10-30-201806:52 AM - edited on 10-15-202106:26 AM by Closed Account
Bhavin Patel1; Penny Jensen1; Leigh Foster1; Renuka Sabnis1; Aaron Gajadhar2; Rosa Viner2; Andreas Huhmer2; Kay Opperman1; John Rogers1
Purpose: Many genetic mutations in cancer cells alter protein expression from AKT, RAS and TP53 pathways. Quantitative measurements of alterations in the expression of pathway proteins and post-translational modifications (PTM) are necessary for classifying disease states, monitoring cancer progression and determining treatment response. Major bottlenecks for quantitation of these proteins are a lack of rigorously verified methods and reagents, and reliance on Western blotting. To address these bottlenecks, we have optimized a multiplex immunoprecipitation (IP) to targeted mass spectrometry (MS) workflow to develop the SureQuantTM pathway panels, thereby achieving simultaneous enrichment and absolute quantitation of multiple total and phospho proteins from the AKT pathway, RAS and TP53.
Methods: The SureQuantTM total and phospho pathway panels contain two modules: 1) The IP and 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 UltimateHeavy and/or AQUA UltimateLight Peptides, and verified MS instrument methods and Skyline software analysis templates. A calibration curve is generated using constant amounts of heavy peptide and variable amounts of light peptide (0.03-200fmol). IP-enriched, digested samples spiked with heavy peptides are analyzed using targeted MS (nanoLC-PRM/MS) and Skyline software.
Results: Previously, we verified antibodies and target peptides to AKT and RAS pathways using an optimized IP-MS workflow. From the standard curve, all target peptides were monitored with <20% CV, 2-3 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 all target peptides was achieved in multiple cancer cell lysate samples with <20% CV between replicates.
1 Thermo Fisher Scientific, Rockford, IL
2 Thermo Fisher Scientific, San Jose, CA