Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3a, 06120, Halle (Saale), Germany.
J Am Soc Mass Spectrom. 2019 Jan;30(1):139-148. doi: 10.1007/s13361-018-1952-8. Epub 2018 Apr 20.
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 °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. Graphical abstract ᅟ.
可裂解交联剂在化学交联/质谱 (MS) 中变得越来越重要，因为它们允许在蛋白质和蛋白质组装体的结构研究中进行可靠和自动化的数据分析。在这里，我们介绍 1,3-二烯丙基脲 (DAU) 作为第一个 CID-MS/MS 可裂解、光硫反应性交联剂。DAU 是一种市售的、廉价的试剂，在存在自由基引发剂的情况下，在 UV-A 照射下，可有效地与半胱氨酸残基发生反 Markovnikov 氢硫化反应。自由基半胱氨酸交联通过正交的“点击反应”进行，并生成稳定的烷基硫醚产物。DAU 在生理 pH 值下反应，并且可以在低至 4°C 的温度下进行肽和蛋白质的交联反应。在串联 MS 实验中，中心脲键在碰撞激活时有效地裂解，产生特征产物离子。这提高了自动交联识别的可靠性。使用含巯基的化合物半胱氨酸和谷胱甘肽，已经筛选了不同的自由基引发剂来进行 DAU 的交联反应。我们的概念也已经通过生物相关蛋白 bMunc13-2 和视网膜鸟苷酸环化酶激活蛋白-2 得到了例证。
