Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid

In traditional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. In this study, we employ an alternative approach using benzoylphenylalanine (BPA), a photoreactive amino acid, incorporated into a disordered region of the human protein HSPB5. BPA is incorporated at specified sites at the time of protein expression, enabling the targeting of any site, including those that are inaccessible to conventional crosslinking reagents. BPA can react with all amino acids, which also overcomes limitations of selective reactivity. However, this broad reactivity imposes additional challenges for crosslink identification. We report and characterize the experimental methods and informatics pipeline used to identify and visualize residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, depending on the site of BPA incorporation. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between technical replicates. Based on these results, we anticipate that this approach will be a powerful, general strategy for characterizing the structures of proteins that have resisted high-resolution characterization, including disordered and heterogeneous proteins.