Figure 4.

Competitive MS-based reactivity profiling defines cysteines liganded by BDHI analogues. (A) Workflow for competitive measurement of cysteine ligandability with fragment electrophiles using IA−DTB probe. Steps include the treatment of cell lysate with compounds (500 μM, 4 h), treatment with IA−DTB probe (500 μM, 2 h), trypsin digestion, TMT labeling, combination, avidin enrichment for biotinylated cysteine-containing peptides, and analysis of competition ratio. (B) Structures and competition ratios (log₂ FC values) from Jurkat cell proteome treated with BDHI 8, 10, and 18. (C) Venn diagram representation of the number of cysteine sites significantly liganded (log₂ FC > 2) by BDHIs. Results were obtained by comparing the site overlap at a given competition ratio threshold for each ligand. (D) Heatmap of competition ratios for representative cysteines and fragments. (E) Crystal structure of HDAC8 (PDB: 2V5W). The zinc binding domain is shown along with reactive cysteines (depicted in magenta). (F) Crystal structure of substrate-bound, human ADA (PDB: 3IAR) with possible cysteine sites for covalent modification. Adenosine is depicted in cyan. (G) Competition binding assay between BDHI 18 and BDHI-alkyne 5. Recombinantly expressed and purified PGK1, GSTP1, and PIN1 were treated with each compound (500 μM, 4 h), labeled with BDHI 5 (500 μM, 2 h), and conjugated with TAMRA-N₃ for in-gel fluorescence scanning.