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

• Published in: Scientific reports 2016-06, Vol.6 (1), p.28107-28107, Article 28107
• Main Authors: Müller, André C., Giambruno, Roberto, Weißer, Juliane, Májek, Peter, Hofer, Alexandre, Bigenzahn, Johannes W., Superti-Furga, Giulio, Jessen, Henning J., Bennett, Keiryn L.
• Format: Article
• Language: English
• Subjects: 631/45/275; 631/45/475; 82; 82/16; 82/58; 96; Adaptor Proteins, Signal Transducing - metabolism; Adenosine triphosphate; Adenosine Triphosphate - analysis; Adenosine Triphosphate - chemistry; Amino Acid Sequence; ATP; Chromatography; Chromatography, High Pressure Liquid; Cytoskeletal Proteins - metabolism; DEAD-box RNA Helicases - chemistry; HEK293 Cells; Humanities and Social Sciences; Humans; Kinases; Kinetics; Mass Spectrometry; multidisciplinary; Oxygen Isotopes - chemistry; Peptides; Peptides - chemistry; Peptides - metabolism; Phosphorylation; Protein Kinases - metabolism; Proteins; Science; Scientific imaging; Signal transduction; Substrate Specificity; Substrates
• ISSN: 2045-2322; 2045-2322
• 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 O 2 ]-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.