Figure 3.

Proteome-wide screening of lysine-reactive fragment electrophiles. a, General protocol for competitive isoTOP-ABPP. Competition ratios, or R values, are measured by quantifying the relative MS1 chromatographic peak intensities for 1-labeled peptides in DMSO- (heavy, or blue) versus fragment-treated (light, or red) samples. An R value of ≥ 4 was used to define a fragment liganding event for a quantified lysine. b, General structures of a lysine-reactive, electrophilic fragment library. See Supplementary Fig. 4 for chemical structures of library members. c, Fraction of total quantified lysines and proteins that were liganded by fragment electrophiles (left panel); of the liganded proteins, the fraction that is found in Drugbank (middle panel); functional classes of liganded Drugbank and non-Drugbank proteins (right panel). d, Number of liganded and quantified lysines per protein. Analysis was applied to proteins containing at least one liganded lysine. e, R values for ten lysines in PFKP, identifying K688 as the only liganded lysine in this protein. Each point represents a distinct fragment-lysine interaction quantified by isoTOP-ABPP. The red dashed line marks an R value of 4 used to define a fragment liganding event. f, Comparison of the ligandability of lysine residues as a function of reactivity with probe 1 (as measured in Fig. 1). Individual lysines are plotted on the x-axis sorted by reactivity, which is shown on the left y-axis, with lower R values correlating with elevated reactivity. A histogram with a bin-size of 200 is shown in blue for the percentage of liganded lysines within each reactivity bin (percent values shown on the right y-axis). g, Lysine reactivity distribution for both liganded and unliganded lysine residues labeled by probe 1. h, Overlap of proteins harboring liganded lysines and liganded cysteines. Cysteine ligandability was taken from reference ¹⁰.