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Screening One-Bead-One-Compound Peptide Libraries for Optimal Kinase Substrates

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Part of the Methods in Molecular Biology book series (MIMB,volume 1360)

Abstract

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 107 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.

Key words

  • Protein kinase
  • Substrate specificity
  • Sequence specificity
  • Peptide library
  • One-bead-one-compound library

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  • DOI: 10.1007/978-1-4939-3073-9_13
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Acknowledgements

The work in this laboratory is supported by the National Institutes of Health (GM062820 and CA0132855).

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Correspondence to Dehua Pei .

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Trinh, T.B., Pei, D. (2016). Screening One-Bead-One-Compound Peptide Libraries for Optimal Kinase Substrates. In: Zegzouti, H., Goueli, S. (eds) Kinase Screening and Profiling. Methods in Molecular Biology, vol 1360. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3073-9_13

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  • DOI: https://doi.org/10.1007/978-1-4939-3073-9_13

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3072-2

  • Online ISBN: 978-1-4939-3073-9

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