Fig. 1

The kinase inhibitor pulldown assay. A Structure of 9 Kinase inhibitors used for KiP. For afatinib, axitinib, AZD4547, and FRAX597, a C3 Linker (yellow line) is added for conjugation. The amine group for conjugation is marked with an asterisk (*). B Workflow for the kinase inhibitor pulldown assay. Native protein lysates are incubated with kinase inhibitor-conjugated beads for 1 h, and non-specific bound proteins are washed with high salt containing buffers. Inhibitor-bound kinases are digested with trypsin overnight, and digested peptides are cleaned with a detergent-removal kit and analyzed by mass spectrometry using a hybrid DDA/PRM mode. C Clustering of protein kinases enriched by individual inhibitors. Single inhibitor bead pulldown was carried out in triplicates for the 6 reference cell line mixture (6REF). Hierarchical clustering analysis of kinases in these experiments clearly shows that each kinase inhibitor pulls down distinct pools of kinases. Kinase classification by illuminating the Druggable Genome (IDG) Target Development Level (IDG-TDL) is indicated with different colors [26]. Green: Tbio, orange: Tchem, blue: Tclin, black: Tdark. FunCats are an in-house annotation of Functional Categories for different kinase targets including lipids (KI-L), metabolite (small molecule) (KI-M), proteins (KI-P) and unknown (KI-X). FunCats mapping can be found in the Additional file 2. D The Kinome tree with identified kinases highlighted. Colors represent IDG TDL classifications, and the size of the circle represents number of inhibitors that can pull down that kinase. Image rendered with KinomeRender [25]. Green: Tbio, orange: Tchem, blue: Tclin, black: Tdark. Original kinome tree illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com)