Abstract
The human genome encodes about 25,000 genes. This number seems to be very small compared to the multitude of different protein functions in highly regulated pathways that are responsible for complex biochemical mechanisms like growth, metabolism, signal transduction and reproduction. Obviously, there are mechanisms creating additional protein diversity. The most important mechanism is post-translational modification (PTM) changing protein surfaces by phosphorylation, sulfation, acetylation, methylation and sumoylation resulting in an about 100-fold higher complexity (1, 2). This chapter presents a very efficient way to detect potential phosphorylation sites in proteins using overlapping peptide scans immobilized on glass slides.
Results from 35 different human kinases using peptide microarrays displaying overlapping peptide scans through either all human cyclophilins or all human FK506-binding proteins are shown. Additionally, detection of phosphorylation sites in a proteome-wide manner is demonstrated using peptide microarrays displaying cytomegalovirus proteome in the form of more than 17,000 overlapping peptides.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Geiss-Friedlander, R., Melchior, F. (2007). Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol. 8, 947–56.
Hunter, T. (2007). The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol Cell. 28, 730–8.
Zhang, H., Zha, X., Tan, Y., Hornbeck, P. V., Mastrangelo, A. J., Alessi, D. R. Polakiewicz, R. D., Comb, M. J. (2002). Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs. J Biol Chem. 277, 39379–87.
Adams, J. A. (2003). Activation loop phosphorylation and catalysis in protein kinases: is there functional evidence for the autoinhibitor model? Biochemistry. 42, 601–7.
Yu, R. K., Bieberich, E. (2001). Regulation of glycosyltransferases in ganglioside biosynthesis by phosphorylation and dephosphorylation. Mol Cell Endocrinol. 177, 19–24.
Bodenmiller, B., Mueller, L. N., Mueller, M., Domon, B., Aebersold, R. (2007). Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods. 4, 231–237.
Kay, J. E. (1996). Structure–function relationships in the FK506-binding protein (FKBP) family of peptidylprolyl cis–trans isomerases. Biochem J. 314, 361–85.
Fanghänel, J., Fischer, G. (2004). Insights into the catalytic mechanism of peptidyl prolyl cis/trans isomerases. Front Biosci. 9, 3453–78.
Drakenberg, T., Forsen, S. (1971). The barrier to internal rotation in monosubstituted amides. J Chem So. Chem Commun. 1404–1405.
Frank, R. (2002). The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports – principles and applications. J Immunol Methods. 267, 13–26.
Reineke, U., Volkmer-Engert, R., Schneider-Mergener, J. (2001). Applications of peptide arrays prepared by the SPOT-technology. Curr Opin Biotechnol. 12, 59–64.
Martin, K., Steinberg, T. H., Cooley, L. A., Gee, K. R., Beechem, J. M., Patton, W. F. (2003). Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye. Proteomics 3, 1244–55.
Steinberg, T. H., Agnew, B. J., Gee, K. R., Leung, W. Y., Goodman, T., Schulenberg, B., Hendrickson, J., Beechem, J. M., Haugland, R. P., Patton, W. F. (2003). Global quantitative phosphoprotein analysis using multiplexed proteomics technology. Proteomics 3, 1128–44.
Zerweck, J., Masch, A., Schutkowski, M. (2009). Peptide microarrays for profiling of modification state specific antibodies, in Epitope mapping protocols, methods in molecular biology (Reineke, U. and Schutkowski, M., eds), Humana, Totowa, NJ, Methods Mol. Biol. 524, 169–80.
Beausoleil. S. A., Jedrychowski, M., Schwartz, D., Elias, J. E., Vilén, J., Li, J., Cohn, M.A., Cantley, L. C., Gygi, S. P. (2004). Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci USA. 101, 12130–5.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Thiele, A., Zerweck, J., Weiwad, M., Fischer, G., Schutkowski, M. (2009). High-Density Peptide Microarrays for Reliable Identification of Phosphorylation Sites and Upstream Kinases. In: Cretich, M., Chiari, M. (eds) Peptide Microarrays. Methods in Molecular Biology™, vol 570. Humana Press. https://doi.org/10.1007/978-1-60327-394-7_9
Download citation
DOI: https://doi.org/10.1007/978-1-60327-394-7_9
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-393-0
Online ISBN: 978-1-60327-394-7
eBook Packages: Springer Protocols