Identification of Direct Kinase Substrates via Kinase Assay-Linked Phosphoproteomics

Methods Mol Biol. 2016:1355:263-73. doi: 10.1007/978-1-4939-3049-4_18.


Protein phosphorylation plays an essential role in the regulation of various cellular functions. Dysregulation of phosphorylation is implicated in the pathogenesis of certain cancers, diabetes, cardiovascular diseases, and central nervous system disorders. As a result, protein kinases have become potential drug targets for treating a wide variety of diseases. Identification of kinase substrates is vital not only for dissecting signaling pathways, but also for understanding disease pathologies and identifying novel therapeutic targets. However, identification of bona fide kinase substrates has remained challenging, necessitating the development of new methods and techniques. The kinase assay linked phosphoproteomics (KALIP) approach integrates in vitro kinase assays with global phosphoproteomics experiments to identify the direct substrates of protein kinases. This strategy has demonstrated outstanding sensitivity and a low false-positive rate for kinase substrate screening.

Keywords: KALIP; Kinase assay; Kinase substrate; Phosphopeptide enrich ment; Phosphoproteomics; pepKALIP; proKALIP.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Chromatography, Affinity
  • Chromatography, Reverse-Phase
  • Computational Biology
  • Databases, Protein
  • Humans
  • Mass Spectrometry
  • Peptide Mapping
  • Phosphopeptides / analysis*
  • Phosphopeptides / chemistry
  • Phosphopeptides / metabolism
  • Phosphorylation
  • Protein Kinases / metabolism*
  • Protein Processing, Post-Translational
  • Proteomics / methods*
  • Substrate Specificity
  • Workflow


  • Phosphopeptides
  • Protein Kinases