A Whole Proteome Inventory of Background Photocrosslinker Binding
A Whole Proteome Inventory of Background Photocrosslinker Binding
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DOI：
10.1002/anie.201605993
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发表时间：
2017-01-24
影响因子：
16.6
通讯作者：
Sieber, Stephan A.
中科院分区：
文献类型：
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作者：
Kleiner, Philipp;Heydenreuter, Wolfgang;Sieber, Stephan A.
基于亲和力的蛋白质谱分析(AfBPP)是一种广泛应用的生物活性分子靶标识别方法。含有光交联剂的探针，如二苯甲酮、二叠氮和芳基叠氮化合物，在紫外光照射下将目标分子不可逆地连接到其目标蛋白质上。尽管它们广泛应用，但人们对光交联剂特定的脱靶作用知之甚少，这影响了结果的可靠性。在此，我们研究了无凝胶定量蛋白质组学标记背景蛋白。识别每个光反应组的特征偏离目标，并汇编成一份综合清单。在一项原理验证研究中，一种蛋白激酶A抑制剂H8被配备了二氮嗪部分。这一光探针的应用揭示了通过与二氮杂环背景的对准，对其原位蛋白质组靶标的前所未有的洞察。综上所述，我们的发现指导了光探针实验中生物相关粘结剂的识别。
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.