Identification of ABL Kinase Substrates by Kinase Assay Linked Phosphoproteomics and Multiple Drug Treatments

The kinase assay linked phosphoproteomics (KALIP) strategy was used to identify direct substrates of the Abelson tyrosine kinase (ABL). First, kinase assays using peptides and proteins from chronic myelogenous leukemia cells and recombinant ABL were done to identify kinase substrates in vitro. The ABL-dependent endogenous phosphoproteome was also examined using multiple drug treatments (imatinib, dasatinib, and bosutinib versus a vehicle treated control) and label-free quantitation after LC-MS/MS. The in vitro and in vivo substrate candidates were then compared to produce a list of highly confident direct ABL substrates.