Screening One-Bead-One-Compound Peptide Libraries for Optimal Kinase Substrates.
Protein kinases phosphorylate specific serine, threonine, and/or tyrosine residues in their target proteins, resulting in functional changes of the target proteins such as enzymatic activity, cellular location, or association with other proteins. For many kinases, their in vivo substrate specificity is at least partially defined by the amino acid sequence surrounding the phosphorylatable residue (or sequence specificity). We report here a robust, high-throughput method for profiling the sequence specificity of protein kinases. Up to 10(7) different peptides are rapidly synthesized on PEGA beads in the one-bead-one-compound format and subjected to kinase reaction in the presence of [γ-S]ATP. Positive beads displaying the optimal kinase substrates are identified by covalently labeling the thiophosphorylated peptides with a fluorescent dye via a disulfide exchange reaction. Finally, the most active hit(s) is identified by the partial Edman degradation-mass spectrometry (PED-MS) method. The ability of this method to provide individual sequences of the preferred substrates permits the identification of sequence contextual effects and non-permissive residues. This method is applicable to protein serine, threonine, and tyrosine kinases.
Department of Chemistry and Biochemistry, The Ohio State University, 578 Biosciences Building, 484 West 12th Avenue, Columbus, OH, 43210, USA.
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Department of Chemistry and Biochemistry, The Ohio State University, 578 Biosciences Building, 484 West 12th Avenue, Columbus, OH, 43210, USA. pei.3@osu.edu.
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D000180:Acrylic Resins; D005456:Fluorescent Dyes; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism*; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000379:methods; D008863:Microspheres; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D011092:Polyethylene Glycols; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020539:Sequence Analysis, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000180:Acrylic Resins; D005456:Fluorescent Dyes; D055767:Immobilized Proteins; D007202:Indicators and Reagents; D019151:Peptide Library; D010455:Peptides; C520358:poly(acryloyl-bis(aminopropyl)polyethylene glycol); D011092:Polyethylene Glycols; D011494:Protein Kinases
One-bead-one-compound library; Peptide library; Protein kinase; Sequence specificity; Substrate specificity