Kinase Assay-Linked Phosphoproteomics: Discovery of Direct Kinase Substrates

Dissection of direct kinase-substrate relationships provides invaluable information about phosphorylation pathways and can highlight both pathogenic mechanisms and possible drug targets for diseases in which abnormal kinase activity is linked to onset and progression. Here, we describe a mass spectrometry-based strategy to define the direct substrates of a kinase of interest. The kinase assay-linked phosphoproteomics approach examines putative kinase substrates both in vitro and in vivo to produce a list of highly confident substrates.