A bead-based approach for large-scale identification of in vitro kinase substrates†

Deciphering the kinase–substrate relationship is vital for the study of phosphorylation network. The use of immobilized proteins on protein chip as the library for screening of potential kinase substrates is a tried-and-tested method. However, information on phosphorylation sites is lacking and the creation of the library with proteins of whole proteome by recombinant expression is costly and difficult. In this study, a new solid-phase approach by immobilization of proteins from cell lysate onto beads as a protein library for kinase substrate screening was developed. It was found that consensus phosphorylation sites motif for kinase substrates could be accurately determined and hundreds of in vitro kinase substrates and their phosphorylation sites could be identified by using this method.