Method for determining protein kinase substrate specificities by the phosphorylation of peptide libraries on beads, phosphate-specific staining, automated sorting, and sequencing

A method is described for the elucidation of the peptide substrate phosphor ylation specificity of a protein kinase. Peptide libraries with two to six degenerate positions and a length of seven or nine amino acids were generat ed directly on Sepharose beads by solid-phase peptide synthesis according t o the split-and-mix procedure. The immobilized peptides were incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase (PKA) as a model enzyme resulting in the phosphorylation of the beads that contain t he recognition motif of the kinase. The beads were then stained with a new phosphate-monoester-specific fluorescent dye consisting of a complex of iro n(III) with fluorescein-coupled iminodiacetic acid. A flow cytometer was us ed to analyze the phosphorylation efficiency and the beads with the highest phosphorylation degree were isolated by the use of a fluorescence-activate d cell sorter. Pool sequencing of those beads revealed the preferred kinase motif. The results are in good agreement with data from the literature. Th e method lends itself to the rapid elucidation of the specificity of unchar acterized protein kinases. (C) 1999 Academic Press.