Abstract LB-177: Quantification of cell signaling proteins by immuno-MALDI

Abstract

Abstract Introduction. To improve cancer patient stratification and overcome the drawbacks of currently used approaches to assess signaling pathway proteins, we set out to develop immuno-matrix assisted laser ionization/desorption (iMALDI) assays combined with a phosphatase-based phosphopeptide quantitation (PPQ) approach to measure PI3K/AKT/mTOR pathway activity. Specifically, we targeted the C-terminal tryptic peptides of AKT1 and AKT2 due to their involvement in full kinase activation. Methods. Following trypsin digestion of cell lysate, stable isotope-labeled standard (SIS) peptides are added. After splitting the solution, one aliquot is treated with phosphatase. Bead-coupled antibodies enrich the non-phosphorylated target peptides, which are washed and spotted onto a MALDI plate, and acidic MALDI matrix elutes the peptides. The resulting light/heavy ratios of both aliquots allow the calculation of protein expression levels and peptide phosphorylation stoichiometry. Results. The iMALDI assays were validated for linear range and accuracy, as well as interference screening, requiring only ~50 µg of total cell or tissue lysate protein to quantify both AKT1 and AKT2 expression levels and phosphorylation stoichiometry. CVs of technical replicates were found to consistently be below 10%. We were able to quantify AKT1 and AKT2 from various cell lines and fresh frozen tumor samples, including SW480 and HCT116 colon cancer cell lines, MDA-MB-231 breast cancer cells, and colon cancer and breast cancer tumor lysates. Further, a direct comparison of matched fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissues showed that the expression levels and phosphorylation stoichiometry quantified differ depending on the tissue preservation method, and that phosphorylation stoichiometries above 30% occur in only a small subset of samples. A direct comparison of four pairs of normal and adjacent tumor tissues showed elevated AKT1 phosphorylation stoichiometry of ~40% in a colorectal cancer liver metastasis, and significantly elevated AKT1 (3.6-fold) and AKT2 (2.2-fold) expression levels in a surgical breast tumor sample. Future directions. In a next step, patient-derived mouse xenograft tissue samples, collected after specific drug treatment, will be analyzed, and the AKT results will be correlated to genomic data to answer the hypothesis whether the AKT expression levels and phosphorylation stoichiometries correlate with response to treatment. Citation Format: Robert Popp, René P. Zahedi, André LeBlanc, Yassene Mohammed, Adriana Aguilar-Mahecha, Oliver Pötz, Mark Basik, Gerald Batist, Christoph H. Borchers. Quantification of cell signaling proteins by immuno-MALDI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-177.