Binding affinities for the two fluorescently-tagged peptides were then determined using a direct fluorescence anisotropy assay (Figure , Supplementary Figure ). Interestingly the binding affinities (Bpa-P1-FAM: 29 ± 10 µM, Bpa-P1-FAM: 14 ± 1 µM) were substantially weaker than is usually observed from a standard RaPID selection, where (sub-)nanomolar affinities are frequently observed. Having established that our peptides were able to bind to BRD3-BD2, we next assessed the crosslinking efficiency of the unlabelled variants. We were pleased to find that both peptides efficiently crosslinked to BRD3-BD2 upon exposure to UV light. After incubation with two equivalents of peptide and irradiation at 365 nm for only 5 minutes, we observed around 40% protein modification as analysed by sample separation on an SDS-PAGE gel and Coomassie staining. As expected, longer UV exposure led to higher protein modification, reaching 96% for Bpa-P1 and 78% for Bpa-P2 after 40 minutes (Figure ). This superior crosslinking ability of Bpa-P1 corresponded well with its higher sequence enrichment in the selection, though it contrasts with the slightly weaker binding affinity we determined. This discrepancy may be explained by the Bpa moiety in Bpa-P1 being more optimally positioned for protein crosslinking than in Bpa-P2. Mass spectrometry was then used to confirm the protein modification observed was due to the addition of a single peptide molecule. Following incubation of BRD3-BD2 with each peptide we observed a mass shift corresponding to addition of a single peptide (Figure ).