USE OF SYNTHETIC PEPTIDE LIBRARIES AND PHOSPHOPEPTIDE-SELECTIVE MASS-SPECTROMETRY TO PROBE PROTEIN-KINASE SUBSTRATE-SPECIFICITY

To search for peptides which serve as substrates for protein kinases, an approach based on peptide libraries has been developed. These pepti de libraries are chemically synthesized by a modified ''divide-couple- recombine'' strategy. After reaction with the kinase of interest, the most highly phosphorylated substrate (selected from the library) is id entified using on-line liquid chromatography-electrospray mass spectro metry (LC-ESMS). Negative ion LC-ESMS with stepped collision energy is used to identify phosphorylated peptides in the enzyme reactions. As predicted, the cAMP-dependent protein kinase is shown to preferentiall y phosphorylate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in a library co nsisting of 19 variants of Kemptide substituted at position 2. Additio nal experiments have been carried out on the nonreceptor tyrosine kina se v-Abl using a peptide library based on the v-Src autophosphorylatio n site -Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly). These result s indicate that Ile is the optimal residue at the position N-terminal to tyrosine. Individual peptides containing the Glu-Asp-Ala-Ile-Tyr mo tif have V-max/K-m values 6-fold higher than the peptide based on the autophosphorylation site itself, confirming the results of the library experiments. This motif has been identified in several tyrosine kinas es at a position in the sequence not previously reported to serve as a phosphorylation or autophosphorylation site.