A 10,000 member PNA-encoded peptide library for profiling tyrosine kinases

A 10,000 member peptide nucleic acid (PNA) encoded peptide library was prepared, treated with the Abelson tyrosine kinase (Abl), and decoded using a DNA microarray and a fluorescently labeled secondary antiphosphotyrosine antibody. A dual-color approach ensured internal referencing for each and every member of the library and the generation of robust data sets. Analysis identified 155 peptides (out of 10,000) that were strongly phosphorylated by Abl in full agreement with known Abl specificities. BLAST analysis identified known cellular Abl substrates such as c-Jun amino-terminal kinase as well as new potential target proteins such as the G-protein coupled receptor kinase 6 and diacylglycerol kinase gamma. To illustrate the generalization of this approach, two other tyrosine kinases, human epidermal growth factor 2 (Her2) and vascular endothelial growth factor receptor 2/kinase insert domain protein receptor (VEGFR2/KDR), were profiled allowing characterization of specific peptide sequences known to interact with these kinases; under these conditions Her2 was demonstrated to have a marked preference for D-proline perhaps offering a unique means of targeting and inhibiting this kinase.