title: Use of synthetic peptide libraries and phosphopeptide-selective mass spectrometry to probe protein kinase substrate specificity.
abstract: To search for peptides which serve as substrates for protein kinases, an approach based on peptide libraries has been developed. These peptide 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 identified using on-line liquid chromatography-electrospray mass spectrometry (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 preferentially phosphorylate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in a library consisting of 19 variants of Kemptide substituted at position 2. Additional experiments have been carried out on the nonreceptor tyrosine kinase v-Abl using a peptide library based on the v-Src autophosphorylation site (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly). These results indicate that Ile is the optimal residue at the position N-terminal to tyrosine. Individual peptides containing the Glu-Asp-Ala-Ile-Tyr motif have Vmax/Km 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 kinases at a position in the sequence not previously reported to serve as a phosphorylation or autophosphorylation site.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook 11794.
authors.forename: J H
authors.initials: JH
authors.lastname: Till
authors.forename: R S
authors.initials: RS
authors.lastname: Annan
authors.forename: S A
authors.initials: SA
authors.lastname: Carr
authors.forename: W T
authors.initials: WT
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis; D010748:Phosphopeptides; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D010455:Peptides; D010748:Phosphopeptides; C037642:kemptide; D011494:Protein Kinases