Analysis of Protein Tyrosine Kinase Specificity Using Positional Scanning Peptide Microarrays

Protein tyrosine kinases phosphorylate their substrates within the context of specific consensus sequences surrounding the site of modification. We describe a peptide microarray approach to rapidly determine tyrosine kinase phosphorylation site motifs. This method uses a peptide library that systematically substitutes each of the amino acid residues at multiple positions surrounding a central tyrosine residue. Peptide substrates are synthesized as biotin conjugates for immobilization on avidin-coated slides. Following incubation of the slide with protein kinase and radiolabeled ATP, the relative extent of phosphorylation of each of the peptides is quantified by phosphor imaging. This method allows small quantities of kinase to be analyzed rapidly in parallel, facilitating analysis of large numbers of kinases.