| description: |
Peptide chips are an emerging technology that could replace many of the bioanalytical methods currently used in drug discovery, diagnostics, and cell biology. The present project focuses on development of a chip strategy for specific screening of protein kinase activities from crude biological extracts and for high-throughput kinetic analysis of novel protein kinase inhibitors. The strategy is based on rational design of specific protein kinase peptide substrates using an electrospray mass-spectrometry based peptide library method, recently developed by the applicant, and on a novel peptide chip technology based of self-assembled monolayers of alkanethiolates on purified gold substrate. The main steps of the project involve determination of substrate specificity profiles for protein kinases selected for validation of the new technology: the isoenzymes of PKC and the calcium calmodulin dependent protein kinase II, and the rational design of novel peptide substrate structures exhibiting the largest degree of selectivity between different kinase forms. The substrates will be synthesised and their kinetic parametres of phosphorylation will be determined for each protein kinase individually. Subsequently, the peptide chips will be generated by immobilisation of the newly designed peptides as microarrays on the monolayer of gold substrate using a selective immobilisation chemistry based on Diels-Alder reaction. The optimisation of the phosphorylation reaction of the peptide microarrays will be performed using purified kinases as well as crude cell extracts, focusing on analysis of several technical parametres including signal intesity, quantitation, background effects and detection limits. |