Abstract 3202: Universal detection of protein phosphorylation based on multi-functionalized soluble nanopolymers

Abstract

Abstract Protein phosphorylation, as the most common covalent modification of proteins, plays a critical role in the regulation of many cellular functions. Irregularities in phosphorylation network are a major cause of onset and progression of many diseases, most notably cancer. Consequently, detection of protein phosphorylation is essential in further understanding of cellular signaling pathways and mechanisms of cell growth and proliferation. To assist in effective phosphorylation analyses, we introduce here a novel technique based on soluble nanopolymers with excellent solubility, compact spherical shape and chemical homogeneity. Soluble nanopolymers functionalized with Ti(IV) ions have shown highly versatility and allow for highly efficient analyses of phosphorylation and signaling pathways. We devise here a novel strategy, termed pIMAGO (phospho-imaging), for effective detection of phosphoproteins on a membrane (Western Blot format) or in a 96-well plate (ELISA format). Unlike antibody-based strategies, the new method is capable of selectively binding a phosphorylated residue independent of amino acid microenvironment. It shows no preference for either of the phosphosites, and thus holds great promise in biological analyses where the site of phosphorylation is not known or its specific antibody is not available. Using this technique, the phosphorylation levels of proteins of interest under physiological conditions can be readily detected as part of a standard Western Blot or ELISA procedures without the need for radioactivity or expensive phosphosite-specific antibodies. The utility of the approach has been demonstrated using standard mixtures of proteins, by in vitro kinase and phosphatase assays, as well as with kinase inhibitor screening and enzyme profiling. In addition, multiplexing capabilities of pIMAGO are presented, a feature often unavailable during antibodies-based detection. Finally, we further confirmed that the technique is sensitive and specific enough to detect endogenous phosphorylation changes by analyzing physiological signaling in number of endogenous protein complexes isolated directly from cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3202. doi:1538-7445.AM2012-3202