A Whole Proteome Inventory of Background Photocrosslinker Binding

被引：92
作者：
Kleiner, Philipp [1 ]
Heydenreuter, Wolfgang [1 ]
Stahl, Matthias [1 ]
Korotkov, Vadim S. [1 ]
Sieber, Stephan A. [1 ]
机构：
[1] Tech Univ Munich, Ctr Integrated Prot Sci, Dept Chem, Lichtenbergstr 4, D-85747 Garching, Germany
来源：
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION | 2017年 / 56卷 / 05期
基金：
欧洲研究理事会;
关键词：
enzymes; photoaffinity labeling; photoreactive probes; protein profiling; proteomics; PHOTOAFFINITY LINKERS; KINASE INHIBITORS; TARGET; PROBES; PROTEINS; H89;
D O I：
10.1002/anie.201605993
中图分类号：
O6 [化学];
学科分类号：
0703 ;
摘要：
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.
引用
收藏
页码：1396 / 1401
页数：6
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