Protocol

Phospho-Proteomics

Volume 527 of the series Methods in Molecular Biology™ pp 269-280

Kinome Profiling Using Peptide Arrays in Eukaryotic Cells

  • Kaushal ParikhAffiliated withDepartment of Cell Biology, University Medical Center Groningen
  • , Maikel P. PeppelenboschAffiliated withDepartment of Cell Biology, University Medical Center Groningen
  • , Tita RitsemaAffiliated withPlant–Microbe Interactions, Institute of Environmental Biology, Utrecht University

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Summary

Over the last 10 years array and mass spectrometry technologies have enabled the determination of the transcriptome and proteome of biological and in particular eukaryotic systems. This information will likely be of significant value to our elucidation of the molecular mechanisms that govern eukaryotic physiology. However, an equally, if not more important goal, is to define those proteins that participate in signalling pathways that ultimately control cell fate. Enzymes that phosphorylate tyrosine, serine, and threonine residues on other proteins play a major role in signalling cascades that determine cell-cycle entry, and survival and differentiation fate in the tissues across the eukaryotic kingdoms. Knowing which signalling pathways are being used in these cells is of critical importance. Traditional genetic and biochemical approaches can certainly provide answers here, but for technical and practical reasons there is typically pursued one gene or pathway at a time. Thus, a more comprehensive approach is needed in order to reveal signalling pathways active in nucleated cells. Towards this end, kinome analysis techniques using peptide arrays have begun to be applied with substantial success in a variety of organisms from all major branches of eukaryotic life, generating descriptions of cellular signalling without a priori assumptions as to possibly effected pathways. The general procedure and analysis methods are very similar disregarding whether the primary source of the material is animal, plant, or fungal of nature and will be described in this chapter. These studies will help us better understand what signalling pathways are critical to controlling eukaryotic cell function.

Key words

Kinase Arrays PepChips Activity Cells Tissues Purified kinases