To further investigate the specificity of the 89 PhABits that crosslinked to BRD4-BD1, the screen was repeated in the presence of the BET inhibitor JQ1, which binds within the acetyl lysine pocket. 46 JQ1 (10 µM) was incubated with hit PhABits (200 µM) and BRD4-BD1 (1 µM) before irradiation and analysis by LC-MS (Figure ). For 68 PhABits, >50% displacement was observed, indicating that these fragments bind selectively to the acetyl lysine binding pocket. Of the remaining 21 fragments that were not displaced, 19 crosslinked to at least three of the other tested proteins, suggesting non-specific crosslinking. Physicochemical analysis of these fragments did not highlight any properties that would typically cause non-specific binding, e.g. high lipophilicity or low solubility (Supplementary Figure ). Therefore, it may be that these PhABits form a long-lived intermediate, leading to diffusion-induced crosslinking. In all subsequent screening analyses these PhABits were omitted.