One of the key challenges of mapping a kinase to particular substrate phosphorylation events is establishing a direct kinase-substrate interaction. Such knowledge would be useful for further understanding signal transduction networks and discovering novel substrates of kinases. Here, we use an analog-sensitive (AS) kinase approach, where we pair a bio-orthogonal ATP analog with a kinase engineered to use this analog. Coupling this approach with stable-isotope labeling and discovery-mode mass spectrometry, we can not only identify kinase substrates but can also quantify their utilization under different contexts, providing fundamental insights into cell signaling pathways.
