Proteomics Analysis of Protein Kinases by Target Class-selective Prefractionation and Tandem Mass Spectrometry

• DOI: 10.1074/mcp.t600062-mcp200
• Publication Date: 2006-12-28T01:12:50Z
• Authors: Josef Wissing; Lothar Jänsch; Manfred Nimtz; Guido Dieterich; Renate Hornberger; György Kéri; Jürgen Wehland; Henrik Daub
• Keywords: Top-down proteomics; Tandem mass tag

Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes eukaryotic cells. However, due to their relatively low abundance compared the vast majority cellular proteins, currently available proteomics techniques do not permit comprehensive biochemical characterization protein kinases. To address these limitations, we have developed prefractionation strategy that uses combination immobilized molecular weight inhibitors for selective affinity capture This approach resulted direct purification cell type-specific sets expressed kinases, and more than 140 different members this enzyme family could be detected by LC-MS/MS. Furthermore enrichment technique combined phosphopeptide fractionation led identification 200 phosphorylation sites on which often remain occluded global phosphoproteome analysis. As states can provide readout signaling activities within system, kinase-selective phosphoproteomics based procedures described here has potential become an important tool transduction