We anticipated that irradiation of a library of photoreactive fragments in the presence of purified protein, would enable photocapture of weak non-covalent fragment-protein binding, which could then be detected by intact mass spectrometry (Figure ). The photocapture event occurs as a two-step process: first, non-covalent fragment-protein binding, characterised by a KD, and second, UV-induced photocrosslinking to capture any reversible interactions, characterised by a crosslinking efficiency. Photoreactive intermediates (e.g. carbenes) rapidly crosslink or are quenched, and therefore photocrosslinking represents a snapshot of the status of the reversible binding equilibrium (Figure ). This contrasts with electrophilic libraries where the warhead has a longer lifetime and the extent of modification is time-dependent. A small alkyl diazirine photoreactive group was selected as the crosslinking moiety to minimise any perturbation to binding of the fragment warhead. A diverse set of 650 amine containing fragments was selected from the GSK amine collection (aromatic ring count ≤2; H-bond donors/acceptors ≤4; heavy atoms ≤15; MW≤200), using clustering on chemical fingerprints (Figure ). A highthroughput synthetic protocol was employed to couple the amine fragments to a carboxylic acid-functionalised diazirine (R1). Reaction mixtures were directly purified by strong cation exchange (SCX) cartridge, followed by deprotection and reverse phase chromatography where required. This workflow enabled the rapid generation of a library of 556 PhABits (Figure ). Analysis of the library's properties confirmed that the fragments covered a chemical space comparable to other commercially available libraries (Supplementary Figure ). Photoreactive fragment screening The PhABit library was screened against a diverse panel of six proteins: myoglobin, lysozyme, carbonic anhydrase, BRD4-BD1, BCL6 and KRas4B G12D . The library was incubated with recombinant protein (1 µM, 4 °C, 15 min, 200 µM PhABit) before irradiation with UV light (302 nm, 10 min), and direct analysis by intact protein LC-MS (Figure ). The resulting mass spectra were analysed to identify PhABits that had covalently modified the protein, leading to an additional peak at a mass of [pro-tein+PhABit-N2]. A range of crosslinking yields (0-53%) were observed and hits were classed as strong (>5% crosslinking) or moderate (1.5-5% crosslinking). In general, the crosslinking yields for hits were low, which could have resulted from either low site occupancy or poor crosslinking due to orientation of the diazirine / quenching of the carbene. Consequently, crosslinking yield was not used as a measure of binding affinity and all hits were followed up. A small number of PhABits (~1%) showed multiple crosslinking events, suggesting that the PhABit was bound to a number of different sites on the protein, or potentially formed a long-lived reactive species enabling multiple modifications to take place via diffusion-based crosslinking. This behaviour was indicative of non-selective binding and therefore not considered hits (Figure ).