New Methods of Phosphoproteomics

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DESCRIPTION (provided by applicant): The reversible process of phosphorylation/dephosphorylation of proteins is a post-translational protein modification that is crucial for intercellular signal transduction. Deregulation of the signal transduction cascade upsets this well balanced system and has been implicated in diseases such as cancer, type II diabetes, cystic fibrosis, Alzheimer's disease, stroke, heart disease and many more. Even though the human genome map presents invaluable insight into the structure and sequence of our genes, it offers limited insight into these critical post-translational protein modifications. Unfortunately, proteomic techniques relevant to the elucidation of signal transduction have been lacking in development in comparison to genomic technologies. The most common technique currently being practiced for the enrichment of phosphorylated substrates is based on the coordination of phosphate groups to metal ion binding sites immobilized on an IMAC (tm) ion exchange resin. Although this approach has been successfully applied to several systems, it is not without experimental limitations and drawbacks in protein selectivity. Our proposed program is aimed at developing an improved method for the efficient and specific immobilization of phosphorylated proteins and peptides. Our approach is based in part on the covalent attachment of phosphorylated groups to the walls of a solid support without the need for metal ion linkages, thus eliminating the complications often encountered in IMAC systems wherein groups other than phosphate complex with the metal ion centers and compromise selectivity. Another unique aspect of our program is focused on the design of organofunctional mesostructured silicas (OMS) for the immobilization of phosphorylated proteins. This support has rigid open framework structures, very high surface areas, and very narrow pore size distributions. These latter features offer important advantages in comparison to resin-based support systems, including, for example, the elimination of the need for specific solvents to swell and access the matrix and the more efficient immobilization of protein by virtue of a higher surface density of functional sites. During the coming funding cycle, we plan to address the following specific aims: 1. Evaluate Mesoporous Immobilized Metal Chromatography (MIMC) as an improvement over the resinbased IMAC approach for the enrichment of phosphorylated substrates through an ion affinity binding mechanism. 2. Develop a phosphopeptide enrichment method using a new solid phase enrichment procedure (SPE) as an improvement over both IMAC and MIMC methodologies. 3. Further advance our new SPE approach to phosphoprotein/peptide enrichment using organofunctional mesostructured silica (OMS) in single bead form as the solid phase host. 4. Evaluate and apply the new technology towards the elucidation the cellular regulation hypoxia inducible factors (HIFs).