(218e) Novel Selective Fluorescent Gel Stains for Improved Detection of Phosphoproteins

The phosphorylation of proteins is critical to regulating cell signaling and ultimately the function of biological systems. Most cellular processes are regulated by reversible phosphorylation of proteins and upwards of 30% of the total complement of proteins expressed by human cells are likely to be phosphorylated at some point during their existence. Determination of protein phosphorylation state is thus important for identifying protein kinase substrates, as well as revealing the on/off state of signal transduction pathways. Selective phosphoprotein staining following polyacrylamide gel electrophoresis is commonly used to identify the potentially phosphorylated protein targets. Staining with Pro-Q Diamond dye, a trivalent cation-loaded calcium ion indicator dye titrated to acidic pH, is one of the most commonly used nonradioactive approaches for such applications. The single commercially available phosphoprotein gel stain is most suitable for a gel scanner equipped with 532 nm laser. This limits the choices of fluorescent gel imagers that can be used for fluorescence-based phosphoprotein analysis. In addition, non-selective staining of non-phosphorylated proteins using the stain is often observed, which makes identification of phosphorylated proteins somewhat challenging. We report on a new family of phosphoprotein stains based upon a highly selective Zn(II) ion chelator operating at neutral pH, which binds to the phosphomonoester residues of phosphoserine, phosphothreonine and phosphotyrosine via a charge-based coordination of the chelated Zn2+ ions. The chelator is readily coupled to a variety of fluorophores, offering the ability to better match the stain with available fluorescent gel imagers, for improved sensitivity readings. The cited dyes were evaluated for selective detection of phosphoproteins in model protein mixes and endogenous phosphoproteins in Jurkat human T cell leukemia cells by either direct gel staining or after transferring the proteins onto a polyvinylidene difluoride membrane support. The dynamic range, sensitivity, and selectivity for detecting phosphoproteins using the new stains matched or exceeded those of the currently available phosphoprotein stain.