Abstract 216: Tracking expression, post-translational modifications and interactions of EGF signalling proteins in A431 cells with antibody microarrays
Authors: Yue Lambert , Pelech Steven 
Published: July 2017

Abstract
Elucidation of epidermal growth factor (EGF) signalling pathways in cancer cells has helped to define the mechanisms for their neoplastic transformation and potential drug targets for therapeutic intervention. Antibody microarrays are promising tools to evaluate alterations in the levels and phosphorylation status of hundreds of proteins of interest with only microgram amounts of crude cell and tissue lysate protein. However, interpretations of the results from traditional antibody microarray approaches have been hampered by the problems associated with sample preparation and protein detection, even when reliable antibodies are deployed in these arrays. The Kinex™ KAM-900P antibody microarray permitted semi-quantitative measurements of the expressions, post-translational modifications and interactions of proteins with 100 µg or less of lysate proteins. These microarrays utilize approximately 878 different pan- and phosphosite-specific antibodies for tracking protein kinases, phosphatases and other low abundance regulatory proteins. Multiple detection protocols were developed with the KAM-900P slides to enable high depth profiling of protein levels, phosphorylation and protein-protein interactions in A431 cells in response to EGF treatment. One method (KAM) involved the capture of in vitro biotin-labeled proteins, followed by their detection with a secondary dye-labeled anti-biotin antibody. False positive signals from associated proteins in complexes with the targets were reduced by chemical cleavage with NTCB prior to their capture on the array, and this also produced more uniformity of the dye signals for protein targets despite vast differences in their sizes. Transient changes in protein phosphorylation in EGF treated cells that were typically lost when processed by conventional methods were better preserved by chemical cleavage right at time of sample homogenization. Biotin-labeling and subsequent detection of the protein on the array with a dye-labeled secondary antibody further reduced non-specific background signals, allowed for a greater dynamic range of detection, and enhanced discrimination of subtle changes. In conjunction with other detection protocols, such as the usage of dye-labeled reporter antibodies for generic protein-tyrosine phosphorylation in sandwich antibody microarrays (SAM format) or generic protein phosphorylation with nanoparticles such as pAMIGO (PAM format), it is also feasible to monitor changes in general post-translational modifications of target proteins or their specific association with other adapter, scaffolding and chaperone proteins of interest for which antibody probes are available.
Citation Format: Lambert Yue, Steven Pelech. Tracking expression, post-translational modifications and interactions of EGF signalling proteins in A431 cells with antibody microarrays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 216. doi:10.1158/1538-7445.AM2017-216