A Whole Proteome Inventory of Background Photocrosslinker Binding

Abstract Affinity‐based protein profiling (AfBPP) is a widely applied method for the target identification of bioactive molecules. Probes containing photocrosslinkers, such as benzophenones, diazirines, and aryl azides, irreversibly link the molecule of interest to its target protein upon irradiation with UV light. Despite their prevalent application, little is known about photocrosslinker‐specific off‐targets, affecting the reliability of results. Herein, we investigated background protein labeling by gel‐free quantitative proteomics. Characteristic off‐targets were identified for each photoreactive group and compiled in a comprehensive inventory. In a proof‐of‐principle study, H8 , a protein kinase A inhibitor, was equipped with a diazirine moiety. Application of this photoprobe revealed, by alignment with the diazirine background, unprecedented insight into its in situ proteome targets. Taken together, our findings guide the identification of biologically relevant binders in photoprobe experiments.