Development of novel technologies for effective phosphorylation analysis

Protein phosphorylation plays a critical role in the regulation of many cellular functions. To assist in phosphoproteomic analyses, in the Tao lab, we have been developing new, more effective techniques based on soluble nanopolymers, which demonstrate excellent solubility, compact spherical shape and chemical homogeneity. Functionalization of such molecules with appropriate groups has demonstrated that the techniques are highly versatile and allow for highly efficient analyses of phosphorylation and signaling pathways. The first dendrimer-based method discussed is PolyMAC (Polymer-based Metal ion Affinity Capture), a phosphopeptide enrichment reagent. Such a reagent is useful because the analysis of phosphoproteomes by mass spectrometry depends on an efficient method to enrich phosphopeptides from complex mixtures. The soluble nature of the novel reagent makes it a competent choice for complete phosphopeptide binding and isolation, which was demonstrated through comprehensive comparisons with existing techniques. The two PolyMAC-based technologies developed were further utilized for biologically-relevant studies of Syk kinase-dependant changes in the breast cancer phosphoproteome and B cell receptor (BCR) stimulated changes in human B cell phosphoproteome. Another approach utilizing nanopolymers we have developed in our lab is designed for effective blotting/detection of phosphoproteins on a membrane or in a 96-well plate. We named this phospho-imaging reagent pIMAGO. Unlike antibodies, the new reagent is capable of selectively binding a phosphorylated residue independent of amino acid microenvironment. Currently, it shows no preference for any of the phosphosites, and thus holds great promise in the 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 easily detected as part of standard Western blot or ELISA formats without the need for radioactivity or expensive phosphosite-specific antibodies. The utility of the approach has been demonstrated using standard mixtures of proteins, as well as by in vitro kinase and phosphatase assays. Overall, dendrimer-based reagents designed for phosphoproteomics have demonstrated outstanding selectivity toward phosphorylated residues, great reproducibility and high recovery yield. They have the potential to become powerful tools for a number of biochemical analyses