Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 56, Issue 5, Pages 1396-1401Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201605993
Keywords
enzymes; photoaffinity labeling; photoreactive probes; protein profiling; proteomics
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Funding
- Deutsche Forschungsgemeinschaft
- CIPSM
- European Research Council (ERC)
- Studienstiftung des deutschen Volkes
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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 kinaseA 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.
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