Having optimised the individual steps of our XL-RaPID methodology, we set out to apply it to a model system. We aimed to identify photocrosslinking peptide ligands for the second bromodomain (BD2) of BRD3 from the bromodomain and extraterminal domain (BET) family of transcriptional coregulators, against which we have recently identified a series of potent non-covalent cyclic peptides. We constructed an mRNA displayed peptide library (>10 12 members) containing six to twelve random amino acids, flanked by an initiator codon and a C-terminal cysteine prior to a GSGSGSlinker. Peptides were initiated with N-chloroacetyl-D-tyrosine (by flexizyme-mediated reprogramming of the start codon), to produce macrocyclic peptides through reaction with the cysteine. Internal positions coding for methionine were reprogrammed to introduce pBpa, resulting in a fraction (approximately 28%) of the library having crosslinking capabilities. We envisaged that through iterative rounds of selection, only sequences containing at least one pBpa would be enriched. The library was then subjected to five rounds of selection against BRD3-BD2 using our optimised XL-RaPID procedure. The peptide library and biotinylated BRD3-BD2 were first exposed to UV light (365 nm) for 30 minutes at 0 °C to promote crosslinking. Following protein capture by magnetic streptavidin beads, stringent 5 M guanidine washes (2 x 20 min) were used to remove ligands that were not covalently linked to the target protein. Finally, the DNA associated with the crosslinked peptides was recovered and carried forward to produce the input library for the next round of selection. We performed five rounds of selection and after observing significant enrichment in round five, next generation sequencing was performed on the recovered DNA libraries (Supplementary Figure , Table ).