A Whole Proteome Inventory of Background Photocrosslinker Binding

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...

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Bibliographic Details
Published in:Angewandte Chemie International Edition 2017-01, Vol.56 (5), p.1396-1401
Main Authors: Kleiner, Philipp, Heydenreuter, Wolfgang, Stahl, Matthias, Korotkov, Vadim S., Sieber, Stephan A.
Format: Article
Language:English
Subjects:
Aromatic compounds
Binders
enzymes
Inventory
Irradiation
photoaffinity labeling
photoreactive probes
protein profiling
Proteins
Proteomics
Target recognition
Ultraviolet radiation
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Summary: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. In affinity‐based protein profiling, small molecules are equipped with a photocrosslinker for irreversible binding to a protein target. The background protein labeling observed with such photocrosslinkers was analyzed by gel‐free quantitative proteomics, and characteristic off‐targets were identified for each photoreactive group. This is of importance for the reliable identification of biologically relevant binders in photoprobe experiments.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.201605993