Identifying direct substrates targeted by protein kinases is important in understanding cellular physiology and intracellular signal transduction. Mass spectrometry-based quantitative proteomics provides a powerful tool for comprehensively characterizing the downstream substrates of protein kinases. This approach is efficiently applied to receptor kinases that can be precisely, directly, and rapidly activated by some agent, such as a growth factor. However, nonreceptor tyrosine kinase Abl lacks the experimental advantage of extracellular growth factors as immediate and direct stimuli. To circumvent this limitation, we combine a chemical rescue approach with quantitative phosphoproteomics to identify targets of Abl and their phosphorylation sites with enhanced temporal resolution. Both known and novel putative substrates are identified, presenting opportunities for studying unanticipated functions of Abl under physiological and pathological conditions.
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