Abstract 5307: Development of pIMAGO for universal detection of protein phosphorylation

• Published in: Cancer research (Chicago, Ill.) Ill.), 2014-10, Vol.74 (19_Supplement), p.5307-5307
• Main Authors: Iliuk, Anton, Li, Li, Pan, Li, Tao, Weiguo A.
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2014-5307

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 in Western Blot 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 procedure without the need for radioactivity or expensive phosphosite-specific antibodies. The utility of the approach has been demonstrated using standard mixtures of proteins and by in vitro kinase and phosphatase assays. 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: Anton Iliuk, Li Li, Li Pan, Weiguo A. Tao. Development of pIMAGO for universal detection of protein phosphorylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5307. doi:10.1158/1538-7445.AM2014-5307