Fig. 4.

K_d chemoproteomic assay with immobilized E-3810. A, schematic view of the experimental design used to calculate the dissociation constant of candidate E-3810 targets for the immobilized drug (K_{d imm}). Light-, medium-, and heavy-labeled proteins are color-coded in black, green, and red, respectively. In the forward experiment, the light protein extract was incubated with E-3810 resin, the medium-labeled lysate was incubated with non-derivatized agarose resin, and the heavy extract was subjected to two subsequent rounds of incubation with the E-3810 resin. After the incubation and washing steps, the resins from the three experiments were mixed to generate light:medium:heavy SILAC triplets. Genuine E-3810 targets are expected to show specificity ratios (M/L in the forward experiment) < 1, whereas the affinity ratios (H/L in the forward experiment) are used to compute the value of K_{d imm E-3810}. B, affinity ranking of the putative kinases targeted by E-3810, based on K_{d imm E-3810}.