A Whole Proteome Inventory of Background Photocrosslinker Binding,Angewandte Chemie International Edition

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A Whole Proteome Inventory of Background Photocrosslinker Binding
Angewandte Chemie International Edition ( IF 16.1 ) Pub Date : 2016-12-16 , DOI: 10.1002/anie.201605993
Philipp Kleiner ₁ , Wolfgang Heydenreuter ₁ , Matthias Stahl ₁ , Vadim S. Korotkov ₁ , Stephan A. Sieber ₁

Affiliation

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.

中文翻译：

背景光交联剂结合的整个蛋白质组清单。

基于亲和力的蛋白质谱分析（AfBPP）是一种广泛用于生物活性分子目标识别的方法。含有光交联剂（如二苯甲酮，重氮嗪和芳基叠氮化物）的探针在受到紫外线照射后，会将目标分子不可逆地连接至其靶蛋白。尽管应用广泛，但对光交联剂特异的脱靶物知之甚少，影响了结果的可靠性。在这里，我们研究了无凝胶定量蛋白质组学对背景蛋白的标记。确定每个光反应性组的特征偏离目标，并将其汇总在综合清单中。在原理验证研究中，H8，一种蛋白激酶A抑制剂装有重氮部分。该光探针的应用表明，通过与二嗪胺的背景相吻合，可以对其原位蛋白质组靶标进行前所未有的洞察。综上所述，我们的发现指导了光探针实验中生物学相关粘合剂的鉴定。

更新日期：2016-12-16

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