Global Incorporation of a Photo-Cross-Linking Amino Acid to Probe the Protein Interactome under Physiological Conditions.

Cross-linking mass spectrometry (XL-MS) has advanced as a powerful approach to map protein-protein interactions in cells. With recent advances in cross-linkers, instrumentation, and software, interactions can be observed in a proteome-wide fashion at residue-level resolution. In most XL-MS experiments, a chemical cross-linker possessing two electrophiles is added to a biological sample and records spatial information by forming covalent bonds between two proximal nucleophilic residues. On the other hand, photo-cross-linking amino acids that can be incorporated into proteins via ribosomal synthesis possess the capacity to rapidly capture transient protein-protein interactions under physiological conditions without introducing exogenous reactive species. Diazirine-based photoamino acids have been widely used for this purpose due to their small size and broad reactivity. Recent studies have shown that diazirines can form MS-cleavable linkages when they react with acidic residues. However, confident interpretation of the resulting spectra and localization of cross-linking sites remain challenging, limiting broader application of diazirine-based approaches in proteome-wide cross-linking analyses. Here, we address this limitation by leveraging DizPK, a diazirine-containing lysine analog with a dedicated MS-cleavable urea functionality. We demonstrate proteome-wide incorporation of DizPK in Escherichia coli via stochastic orthogonal recoding of translation and subsequent identification of photo-cross-linked peptides to map protein-protein interactions under physiological conditions. We find that the resulting approach can provide high-resolution structural information associated with transient biological processes.