title: Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry.
abstract: Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis.
authors.affiliation: Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Wissing
authors.forename: Lothar
authors.initials: L
authors.lastname: Jänsch
authors.forename: Manfred
authors.initials: M
authors.lastname: Nimtz
authors.forename: Guido
authors.initials: G
authors.lastname: Dieterich
authors.forename: Renate
authors.initials: R
authors.lastname: Hornberger
authors.forename: György
authors.initials: G
authors.lastname: Kéri
authors.forename: Jürgen
authors.initials: J
authors.lastname: Wehland
authors.forename: Henrik
authors.initials: H
authors.lastname: Daub
mesh_terms: D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D006801:Humans; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases