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High-throughput Screening of Substrates for Tyrosine Kinases Using PEPscreen Custom Peptide®
Danhui Wang, Fan Zhang, Tao Zhao, Fei Zhong, and Keming Song
Sigma Life Science
Sponsored by Sigma-Aldrich
Published in March 2011 2011 Sponsored,vendor-submitted protocol
Protein Tyrosine Kinases (PTKs) play important roles in modulating a wide variety of cellular events, including differentiation, proliferation, metabolism and apoptosis. Those regulations are mediated by a series of signal cascades that involve phosphorylation of tyrosine residues in target proteins. Therefore, identification of the substrates for PTKs is very important for the studies in both basic research and drug development environment. Here, we report a high-throughput ELISA-based method to identify peptide substrates for class-specific and/or enzyme-specifi c PTKs that can be utilized for the detection of kinase activity both in vivo and in vitro. The amino acid sequences of peptide library containing 13-mer tyrosine peptides were generated by a predicting algorithm developed in-house for all potential protein substrates of PTKs identified from the public databases. Next, the peptides were synthesized using PEPscreen®, a proprietary peptide synthesis platform, and then subjected to a screening of 39 PTKs using an ELISA-based method. The number of peptides selected for each PTK ranges from 2 to 71 with an average of 25 from a total of 376 peptides tested. The 173 sequences selected were classified into different groups with the reactivity to single, multiple, or all PTKs. The reactivity and specificity of the selected peptide substrates were further validated. The validation results indicated that this screening method has a very high sensitivity and reproducibility. Thus, the combination of our algorithm for selecting peptide sequences, the PEPscreen peptide synthesis platform, and ELISA-based assay method provide a successful high-throughput system for the screening of peptide substrates for many PTKs.
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