A second feature of covalent crosslinking is that it enables determination of the site of binding without recourse to crystallography. The site of binding can be determined either by competition, where suitable ligands with known binding location are available (e.g. JQ1 as demonstrated above), or by elucidation of the site of crosslinking by LC-MS/MS analysis. This latter approach would be particularly powerful for proteins without known binders or crystallography. PhABit 2 was selected to explore this technique with BRD4-BD1. Crosslinked samples were digested using trypsin/LysC and the resulting peptides analysed by LC-MS/MS. The peptide 101PGD*DIVLMAEALEK114 was identified as carrying a modification on D103, which was not observed in the non-irradiated control sample (Figure ). This residue is at the edge of the acetyl lysine binding site, and ideally situated for crosslinking.