In this project supported by the Chemical Measurement and Imaging program (with co-funding from the Office of International Science and Engineering), Professor Daniel Talham and coworkers at the University of Florida are exploring metal phosphate and metal phosphonate solids and thin films as a new class of phosphopeptide separation and enrichment media. The expected improvements in performance are based on a new mechanism for phosphopeptide affinity involving the ability of some metal phosphate solids to selectively form covalent linkages to divalent phosphate groups while excluding other common anions. The project will explore new particle systems for affinity enrichment and new surface modification approaches to extend metal phosphonate-based enrichment strategies to on-target techniques. The new mechanisms and materials will be evaluated using surface analytical methods to quantify the activity of the new materials toward different phosphopeptide structures. Finally, newly developed systems will be tested to prove their applicability to phosphoprotein mass spectrometry.
These experiments seek to devise new means of isolating a wide range of biomaterials that are important in high-technology applications ranging from sensing and diagnostics to enabling fundamental studies of proteins - a fundamental building block of life. In addition, training related to biomaterials, interfaces, chemical measurement, and protein chemistry will provide graduate and undergraduate students with the skills and knowledge needed to be competitive in biotechnology related high-technology professions. The project also fosters collaboration with international partners at the Université de Nantes in France. This cooperation includes an extensive international educational component through the exchange of graduate student researchers. These exchanges are designed to facilitate thesis work and to also provide students with important alternative perspectives on research and approaches to scientific exploration.
Phosphorylation is one of the most common post translational modifications of proteins, present in perhaps a third of mammalian proteins at any one time. Protein phosphorylation is associated with a number of regulatory mechanisms such as gene expression, signal transduction, enzyme activity, and cell division and changes in native phosphorylation states are implicated in several diseases. Quantifying phosphorylation and identifying phosphorylation sites is an important requirement for understanding many biochemical pathways and diseases, although phosphorylation is generally substoichiometric, resulting in low abundance of phosphorylated peptides, making their identification difficult. Therefore, it is necessary to couple phosphopeptide enrichment procedures with high sensitivity analytical tools to enable both fundamental studies and faster, more accurate diagnostics. The current project explores metal phosphate and metal phosphonate solids and thin films as a new class of phosphopeptide enrichment media. This NSF-funded project determined that a unique enrichment mechanism is in effect on zirconium phosphate and phosphonate interfaces. Instead of an electrostatic attraction between a positively charged surface and anionic residues on the adsorbate, phosphate groups can form a coordinate covalent bond to the Zr4+. In aqueous conditions, the surface does not possess naked Zr4+ sites, so this mechanism requires displacing the ligands that are present, oxide and hydroxide. Herein lays the specificity of phosphate and phosphonate in these materials. The divalent phosphate is capable of displacing oxide and hydroxide to form covalent linkages to the Zr4+ ions, similar to an IMAC mechanism. Importantly, however, neither carboxylate nor monovalent phosphodiesters are basic enough to displace oxide and hydroxide, so they can only form nonspecific electrostatic bonds to the oxide-like surface, similar to the MOAC mechanism. The principal outcome of the project is validating this mechanism of phosphopeptide selectivity at zirconium phosphate surfaces. The mechanism of phosphopeptide binding to zirconium phosphonate or zirconiumphosphate surfaces can be extended to phosphoproteins and the project also explored using this linkage as a method for immobilizing proteins for array or "protein chip" technologies. For this, a short phosphorylatable peptide sequence, or tag, was developed for fusion into the protein. Upon phosphorylation, the proteins can be easily immobilized onto zirconated glass slides in a single step. The proteins were shown to retain their function. The important outcome is the development and demonstration of a new method for immobilizing proteins for use in analytical or bioanalytical procedures.
