Investigation of Phophorylation Profiles in Cell Lysates or Tumor Homogenates Using Peptide Microarrays

Dr. Rob Ruijtenbeek,Head of Proteomics Research, PamGene, speaking at the OncoProteomics World Congress 2006.

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Abstract
Protein tyrosine kinases are a very important family of enzymes involved in cell signaling. Signals are propagated by phosphorylation of tyrosine, serine and threonine residues in target proteins.

We have developed a peptide microarray assay in which up to 400 different target protein-derived peptides are immobilized on a chip.

One microarray is the size of a single microtitre plate well, and can be used both in a 4-array as well as a 96-array format. Kinase activity present in a sample incubated on an array results in peptide dependant phosphorylation detected by fluorescence.

The unique features of this technology are the convenient mix-and-measure format and the kinetic readout, enabled by the porous nature of the array surface. In a single 96-array experiment several thousand kinetic phorphorylation curves are generated in parallel.

This is a convenient and powerful method for investigating kinase activity profiles in cell lines or tumor tissue homogenates. We have used this assay format to investigate, among others, the effect of kinase inhibitors on kinase activity profiles.

Finally, the alteration of kinase activity in cells after physiological stimulation has been investigated. These microarray based applications in kinase research and their relevance for drug discovery in oncology will be discussed.

About the speaker
Dr. Rob Ruijtenbeek is Head of Proteomics Research within Pamgene (‘s-Hertogenbosch, The Netherlands), where he performs research onmicroarray applications in kinase research and other proteomics areas.

Prior to joining PamGene he worked in the area of medicinal chemistry andasthma research at the faculty of Pharmacy of the University of Utrecht,The Netherlands, where he received his Ph.D. degree in 2001. He holds an M.Sc.degree in biochemistry and organic chemistry from the University of Nijmegen(1996).

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