Kinase-Catalyzed Biotinylation to Map Cell Signaling Pathways: Application to Epidermal Growth Factor Signaling.

Cell signaling involves a network of protein-protein interactions and post-translational modifications that govern cellular responses to environmental cues. To understand and ultimately modulate these signaling pathways to confront disease, the complex web of proteins that becomes phosphorylated after extracellular stimulation has been studied using mass spectrometry-based proteomics methods. To complement prior work and fully characterize all phosphorylated proteins after the stimulation of cell signaling, we developed K-BMAPS (kinase-catalyzed biotinylation to map signaling), which utilizes ATP-biotin as a kinase cosubstrate to biotin label substrates.

Chitosan-assisted permeabilization of ATP-biotin for live cell kinase-catalyzed biotinylation.

Kinases are essential cell signaling enzymes that phosphorylate protein substrates using ATP as the universal cosubstrate. A wide variety of ATP analogs have been used in kinase research, although the studies are limited by the cell impermeability of ATP. Here we describe the use of the cationic polymer deacetylated chitosan to permeabilize ATP analogs for live cell applications, including kinase-catalyzed biotinylation.