The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies
The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies
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DOI：
10.1007/s13361-018-1952-8
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发表时间：
2019-01-01
影响因子：
3.2
通讯作者：
Sinz, Andrea
中科院分区：
文献类型：
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作者：
Iacobucci, Claudio;Piotrowski, Christine;Sinz, Andrea
可裂解的交联剂对于化学交联/质谱（MS）越来越重要，因为它们允许在蛋白质和蛋白质组装的结构研究中进行可靠和自动化的数据分析。在这里，我们引入1，3-二烯丙基脲（DAU）作为第一个CID-MS/MS-可裂解的光硫醇反应性交联剂。DAU是一种市售的廉价试剂，其在UV-A照射时在自由基引发剂的存在下与半胱氨酸残基有效地进行抗马尔可夫氢硫醇化。自由基半胱氨酸交联通过正交点击反应进行，并产生稳定的烷基硫醚产物。DAU在生理pH下反应，并与肽发生交联反应，蛋白质可在低至4 ℃的温度下进行。在串联MS实验过程中，中心脲键在碰撞活化后有效裂解，产生特征产物离子。这提高了自动交联识别的可靠性。不同的自由基引发剂已被筛选用于使用含巯基化合物半胱氨酸和谷胱甘肽的DAU的交联反应。我们的概念也被生物相关蛋白bMunc 13 -2和视网膜鸟苷酸环化酶激活蛋白-2举例说明。
Cleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal click reaction and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 degrees C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2.