Identifying Kinase Substrates via a Heavy ATP Kinase Assay and Quantitative Mass Spectrometry

Sci Rep. 2016 Jun 27;6:28107. doi: 10.1038/srep28107.


Mass spectrometry-based in vitro kinase screens play an essential role in the discovery of kinase substrates, however, many suffer from biological and technical noise or necessitate genetically-altered enzyme-cofactor systems. We describe a method that combines stable γ-[(18)O2]-ATP with classical in vitro kinase assays within a contemporary quantitative proteomic workflow. Our approach improved detection of known substrates of the non-receptor tyrosine kinase ABL1; and identified potential, new in vitro substrates.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism*
  • Adenosine Triphosphate / analysis*
  • Adenosine Triphosphate / chemistry*
  • Amino Acid Sequence
  • Chromatography, High Pressure Liquid
  • Cytoskeletal Proteins / metabolism*
  • DEAD-box RNA Helicases / chemistry
  • HEK293 Cells
  • Humans
  • Kinetics
  • Mass Spectrometry*
  • Oxygen Isotopes / chemistry
  • Peptides / chemistry
  • Peptides / metabolism
  • Phosphorylation
  • Protein Kinases / metabolism
  • Substrate Specificity


  • ABI1 protein, human
  • Adaptor Proteins, Signal Transducing
  • Cytoskeletal Proteins
  • Oxygen Isotopes
  • Oxygen-18
  • Peptides
  • Adenosine Triphosphate
  • Protein Kinases
  • DDX3X protein, human
  • DEAD-box RNA Helicases