Application of zirconium phosphate/phosphonate surfaces in phosphopeptide enrichment and protein immobilization

Phosphorylation is one of the most significant post-translational modifications of proteins in eukaryotes, and is involved in numerous cellular events. Therefore the identification and quantification of phosphorylation sites in proteins is of great importance. With its rapid development, mass spectrometry (MS) appears to be the most powerful technology in proteome research. However, the quantitative characterization of phosphoprotein is still a challenging task because of the low abundance of phosphoprotein, the substoichiometric nature of phosphorylation, and some technical limitations, which makes the isolation and concentration of phosphopeptides prior to MS analysis necessary. To date, ion metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) are the most commonly used methods. However, nonspecific binding from the acidic side chains of nonphosphorylated peptides results in low specificity and sensitivity in both.;The use of zirconium alkyl-phosphonate (ZrP) for capturing molecules containing phosphate groups was first reported by our group and collaborators in studies including DNA or protein immobilization. The first study described in this thesis applied the ZrP surface in phosphopeptide enrichment by first studying the pH influence on peptide binding and then investigating the ability of the surface in distinguishing phosphopeptide analogs, using surface plasmon resonance enhanced ellipsometry (SPREE) technique.;The second project aims to compare the enrichment efficiency of the ZrP surface to TiO2, which is the most widely used material in MOAC. A solution deposition method to grow a TiO2 thin film on gold slides was tried and atomic layer deposition (ALD) was also performed. Four peptides used in project 1 were chosen for the enrichment efficiency comparison using the SPREE technique.;The objective of the third project is to develop a phosphorylatable protein tag for protein immobilization. Five peptide candidates were chosen to evaluate the binding affinity to Zr-phosphate and Zr-phosphonate surfaces. The final chosen peptide was fused with a protein to test its ability to immobilize the protein. A bench prepared amine slide was also tested in the same experiments.;With SPREE and fluorescence techniques, we can obtain mass quantitative information and details on kinetic binding. Other biophysical techniques such as atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) were applied to characterize the surface.