Summary
The reversible phosphorylation of serine, threonine, and tyrosine residues is one of the most important intracellular post-translational modifications regulating enzymatic activities and protein/protein interaction in eukaryotic cells. Tools for determining phosphorylation status of proteins and peptides play a prominent role in signal transduction research and proteomics. Pan-specific antibodies claimed to recognize modified amino acid residues independent on the nature of surrounding residues in peptides and proteins are widely used. We used high-content phosphopeptide microarrays and microarrays displaying acetyllysine-containing peptides for comprehensive characterization of commercially available generic anti-phosphopeptide and anti-acetyllysine antibodies. We were able to demonstrate distinct subsite specificity and cross-reactivity for such antibodies.
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References
Manning, G., Whyte, D. B., Martinez, R., Hunter, T., and Sudarsanam, S. (2002). The protein kinase complement of the human genome. Science 298, 1912–1934.
Kim, S. C., Sprung, R., Chen, Y., Xu, Y., Ball, H., Pei, J., Cheng, T., Kho, Y., Xiao, L., Grishin, N. V., White, M., Yang, X.-J., and Zhao, Y. (2006). Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol. Cell 23, 607–618.
Iwabata, H., Yoshida, M., and Komatsu, Y. (2005). Proteomic analysis of organ-specific post-translational lysine-acetylation and –methylation in mice by use of anti-acetyllysine and –methyllysine mouse monoclonal antibodies. Proteomics 5, 4653–4664.
Kaufmann, H., Bailey, J. E., and Fussenegger, M. (2001). Use of antibodies for detection of phosphorylated proteins separated by two-dimensional gel electrophoresis. Proteomics 1, 194–199.
Salomon, A. R., Ficarro, S. B., Brill, L. M., Brinker, A., Phung, Q. T., Ericson, C., Sauer, K., Brock, A., Horn, D. M., Schultz, P. G., and Peters, E. C. (2003). Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proc. Natl. Acad. Sci. U S A 21, 443–448.
Steinberg, T. H., Agnew, B. J., Gee, K. R., Leung, W. Y., Goodman, T., Schulenberg, B., Hendrickson, J., Beechem, J. M., Haugland, R. P., and Patton, W. F. (2003). Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology. Proteomics 3, 1128–1144.
Craig, A. L., Bray, S. E., Finlan, L. E., Kernohan,N. M., and Hupp, T. R. (2003). Signaling to p53: The use of phospho-specific antibo-dies to probe for in vivo kinase activation. Methods Mol. Biol. 234, 171–202.
Wenschuh, H., Volkmer-Engert, R., Schmidt, M., Schulz, M., Schneider-Mergener, J., and Reineke, U. (2000). Coherent membrane supports for parallel microsynthesis and screening of bioactive peptides. Biopolymers (Peptide Science) 55, 188–206.
Panse, S., Dong, L., Burian, A., Carus, R., Schutkowski, M., Reimer, U., and Schneider-Mergener, J. (2004). Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays. Mol. Divers. 8, 291–299.
Reineke, U., and Schutkowski, M. (2006). Peptide arrays in proteomics and drug discovery, in BioMEMS and Biomedical Nanotechnology, Volume II: Micro and Nano-Technologies for Genomics and Proteomics, (Ozkan, M., and Heller, M. J., eds), Springer Science + Business Media, LLC, New York, pp. 161–282.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Zerweck, J., Masch, A., Schutkowski, M. (2009). Peptide Microarrays for Profiling of Modification State-Specific Antibodies. In: Schutkowski, M., Reineke, U. (eds) Epitope Mapping Protocols. Methods in Molecular Biology™, vol 524. Humana Press. https://doi.org/10.1007/978-1-59745-450-6_12
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DOI: https://doi.org/10.1007/978-1-59745-450-6_12
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