Screening kinase phosphorylation motifs using Peptide libraries.

INTRODUCTIONThis protocol describes a method for oriented peptide library screening. This method, combined with bioinformatics-based searches of protein sequence databases, provides a strategy for identifying substrates of particular protein kinases, including low-abundance proteins. In oriented peptide library screening, a very large number of peptides are synthesized simultaneously by solid-phase peptide synthesis, with all of the peptides containing either a single fixed Ser, Thr, or Tyr residue at an "orienting" position within the peptide sequence. The orienting residue in the collection of peptides serves as the phospho-acceptor during an in vitro phosphorylation reaction with the protein kinase of interest and is flanked by a series of degenerate positions, which contain a mixture of all possible amino acids. Only those peptides that contain favorable amino acids surrounding the fixed Ser, Thr, or Tyr residues will be preferentially phosphorylated by the kinase of interest. This subset of phosphorylated peptides can then be separated from the bulk of nonphosphorylated peptides using immobilized metal affinity chromatography (IMAC). The recovered phosphopeptides are sequenced in bulk by Edman degradation. By comparing the amount of each amino acid at each position flanking the fixed Ser, Thr, or Tyr in the phosphorylated peptides with the amount of each amino acid in the starting peptide library mixture, the affinity of the kinase for each amino acid in each position in the sequence is revealed. The end result of this process is a matrix of selection values that describes, in quantitative terms, the relative importance of each amino acid at each position within the kinase substrate motif, as well as the optimal peptide substrate sequence for that kinase. The matrix of kinase selectivity values can be used to search protein sequence databases and identify potential substrate proteins that contain the closest matches to the optimal protein kinase phosphorylation motif. These putative substrates can then be examined in vitro and in vivo to determine whether they are, in fact, kinase substrates under physiological conditions.