Summary
In view of the importance of information transfer mediated throughout the cell by recognition, phos-phorylation or dephosphorylation of kinases, their adapters, or substrates, this method was developed. The method provides a potent research tool for rapidly generating and testing these substrates as modeled by synthetic peptide arrays. The peptides or phosphorylated peptides are automatically generated on the inner surfaces of microplate wells, covalently linked to a polylysine polymer so that they are in a sterically favorable conformation, immediately available for in situ testing. Products up to 18 amino acids long have shown excellent mass spectral homogeneity. Thus, determinate peptide libraries can be ready for testing in as little as 2 days after the conception of an experiment. The process can be easily automated using robotic liquid handlers and is extremely rapid, sensitive, and economical. Optionally, the method can be upgraded to a higher throughput level using more powerful workstations with greater capacity, such as the Biomek FX, or any similar robotics capable of transfer-from-file logic to guide synthesis cycles.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Manning, B. D., Cantley, L. C. (2002) Hitting the target: emerging technologies in the search for kinase substrates. Sci. STKE 2002, PE49.
Songyang, Z., Cantley, L. C. (1995) Recognition and specificity in protein tyrosine kinase-mediated signalling. Trends Biochem. Sci. 20, 470–475.
Songyang, Z., Carraway, K. L. III, Eck, M. J., Harrison, S. C., Feldman, R. A., Moham-madi, M. et al. (1995) Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature 373, 536–539.
Till, J. H., Annan, R. S., Carr, S. A., Miller, W. T. (1994) Use of synthetic peptide libraries and phosphopeptide-selective mass spectrometry to probe protein kinase substrate specificity. J. Biol. Chem. 269, 7423–7428.
Geysen, H. M., Meloen, R. H., Barteling, S. J. (1984) Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA. 81, 3998–4002.
Gausepohl, H., Boulin, C., Kraft, M., Frank, R. W. (1992) Automated multiple peptide synthesis. Pept. Res. 5, 315–320.
Frank, R. (1992) Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron. 48, 9217–9232.
Tegge, W., Frank, R., Hofmann, F., Dost-mann, W. R. (1995) Determination of cyclic nucleotide-dependent protein kinase substrate specificity by the use of peptide libraries on cellulose paper. Biochemistry 34, 10569–10577.
Tegge, W. J., Frank, R. (1998) Analysis of protein kinase substrate specificity by the use of peptide libraries on cellulose paper (SPOT-method).Methods Mol. Biol. 87, 99–106.
Luo, K., Zhou, P., Lodish, H. F. (1995) The specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library. Proc. Natl. Acad. Sci. USA. 92, 11761–11765.
Saxinger, C. (2000) Automated peptide design and synthesis. United States Patent 6,031,074 (February 29, Issue Date).
Rosenfeld, S. J., Young, N. S., Alling, D., Ayub, J., Saxinger, C. (1994) Subunit interaction in B19 parvovirus empty capsids. Arch. Virol. 136, 9–18.
Kim, P. J., Sakaguchi, K., Sakamoto, H., Saxinger, C., Day, R., McPhie, P. et al. (1998) Colocalization of heparin and receptor binding sites on keratinocyte growth factor. Biochemistry. 37, 8853–8862.
Saxinger, C. (2003) Polypeptides comprising IL-6 ligand-binding receptor domains. US PATENT 6,664,374 (December 16, 2003, issue date).
Saxinger, C. (2007) Polypeptides that bind HIV gp120 and related nucleic acids, antibodies, compositions, and methods of use. United States Patent 7,304,127 (December 4, 2007, issue date).
Saxinger, C., Conrads, T. P., Goldstein, D. J., Veenstra, T. D. (2005) Fully automated synthesis of (phospho)peptide arrays in microtiter plate wells provides efficient access to protein tyrosine kinase characterization. BMC Immunol. 6, 1.
Hudecz, F., Szekerke, M. (1985) Synthesis of new branched polypeptides with poly(lysine) back bone. Collection Czechoslovak Chem. Commun. 50, 103–113.
Mezo, G., Kajtar, J., Hudecz, F., Szekerke, M. (1993) Carrier design – conformational studies of amino acid(X) and oligopeptide (X-Dl-Ala(M)) substituted poly(L-Lysine). Biopolymers33, 873–885.
Chao, H. G., Leiting, B., Reiss, P. D., Bur-khardt, A. L., Klimas, C. E., Bolen, J. B. et al. (1995) Synthesis and applications of Fmoc-O -[bis(dimethylamino)phosphono]-tyrosine, a versatile protected phosphotyrosine equivalent. J. Org. Chem. 60, 7710–7711.
Acknowledgments
I especially thank Paul Nagel and Greg Goetz for shaping the software architecture of the robotic interface and control and Beckman Instruments for providing the file structure of the Biomek 1,000 and Biomek 2,000 array files. I also thank Mei-Wan Ho, Jamshed Ayub, and Vasu Parekh for developmental laboratory assistance and Robert Gallo for support.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Saxinger, W.C. (2009). Protein Tyrosine Kinase Characterization Based on Fully Automated Synthesis of (Phospho) Peptide Arrays in Microplates. In: Graauw, M.d. (eds) Phospho-Proteomics. Methods in Molecular Biology™, vol 527. Humana Press. https://doi.org/10.1007/978-1-60327-834-8_19
Download citation
DOI: https://doi.org/10.1007/978-1-60327-834-8_19
Publisher Name: Humana Press
Print ISBN: 978-1-60327-833-1
Online ISBN: 978-1-60327-834-8
eBook Packages: Springer Protocols