Methods in Molecular Biology Volume 795, 2012, pp 179-190
Date: 25 Aug 2011

Covalent Cross-Linking of Kinases with Their Corresponding Peptide Substrates

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Protein phosphorylation represents the most dominant and evolutionary conserved posttranslational modification for information transfer in cells and organisms. The human genome encodes >500 protein kinases, and thousands of phosphorylation sites are present in mammalian proteome. To develop a global view of phosphorylation network, there is a need to map the connectivity between kinases and phosphoproteome. We developed a chemical kinase–substrate cross-linker 1 that converts transient kinase–substrate interactions into a covalently linked kinase–substrate complex in vitro and in the presence of cell lysates. The method can be applied to identify unknown upstream kinases responsible for phosphorylation events in cell lysates.