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.
Highlights
Protein kinases constitute a large superfamily of enzymes with key regulatory functions in most signal transmission processes of eukaryotic cells
The established technique is further compatible with phosphoproteomics applications, and more than 200 phosphorylation sites from protein kinases could be assigned in this study
Strategy—Because previously described kinase purification methods have used immobilized, ATP-competitive inhibitors as selective ligands for affinity chromatography with some success, we reasoned that further method optimization and the combined use of inhibitor matrices with distinct selectivity profiles might permit the enrichment of a substantial fraction of cellular protein kinases [12, 14, 15, 18, 19]
Summary
The only biochemical techniques permitting the efficient isolation of certain subfractions of the expressed kinome use immobilized low molecular weight kinase inhibitors as selective capture molecules for the affinity enrichment of their cellular target proteins [11,12,13]. We report the development of a chromatographic method using four different kinase-selective affinity resins with distinct target specificities. This fractionation procedure efficiently isolates a large variety of protein kinases from total cell extracts and permitted the detection of up to 113 protein kinases per cell line by LC-MS/MS analysis. The established technique is further compatible with phosphoproteomics applications, and more than 200 phosphorylation sites from protein kinases could be assigned in this study
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