Covalent Cross-Linking of Kinases with Their Corresponding Peptide Substrates

  • Alexander V. Statsuk
  • Kevan M. ShokatEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 795)


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.

Key words

Kinase Substrate Kinase inhibitor Thiophene-2,3-dialdehyde Covalent cross-link 



KMS thanks NIH RO1-EB001987 for funding as well as a grant from Eli Lilly. The authors thank Beatrice Wang for helpful comments on the manuscript. Dustin Maly and Megan Riel-Mehan are acknowledged for their contributions to the project.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of ChemistryNorthwestern UniversityEvanstonUSA
  2. 2.Department of Cellular and Molecular PharmacologyUniversity of California San FranciscoSan FranciscoUSA

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