Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120, Halle/Saale, Germany.
Angew Chem Int Ed Engl. 2017 Nov 13;56(46):14551-14555. doi: 10.1002/anie.201708273. Epub 2017 Oct 9.
Combining the properties of a zero-length cross-linker with cleavability by tandem mass spectrometry (MS/MS) poses great advantages for protein structure analysis using the cross-linking/MS approach. These include a reliable, automated data analysis and the possibility to obtain short-distance information of protein 3D-structures. We introduce 1,1'-carbonyldiimidazole (CDI) as an easy-to-use and commercially available, low-cost reagent that ideally fulfils these features. CDI bridges primary amines and hydroxy groups in proteins with the lowest possible spacer length of one carbonyl unit (ca. 2.6 Å). The cross-linking reaction can be conducted under physiological conditions in the pH range between 7.2 and 8. Urea and carbamate cross-linked products are cleaved upon collisional activation during MS/MS experiments generating characteristic product ions, greatly improving the unambiguous identification of cross-links. Our innovative analytical concept is exemplified and applied for bovine serum albumin (BSA), wild-type tumor suppressor p53, an intrinsically disordered protein, and retinal guanylyl cyclase activating protein-2 (GCAP-2).
将零长度交联剂的特性与串联质谱（MS/MS）的可裂解性相结合，为使用交联/MS 方法进行蛋白质结构分析带来了巨大的优势。这些优势包括可靠的、自动化的数据分析以及获取蛋白质 3D 结构短程信息的可能性。我们引入 1,1'-碳二亚胺（CDI）作为一种易于使用且商业上可获得的、低成本试剂，它理想地满足了这些特性。CDI 在生理条件下将蛋白质中的伯胺和羟基桥接起来，其间隔基长度最短，为一个羰基单元（约 2.6 Å）。在 pH 值为 7.2 到 8 之间的范围内，交联反应可以在生理条件下进行。在 MS/MS 实验中，通过碰撞激活可以裂解脲和氨基甲酸盐交联产物，产生特征性的产物离子，极大地提高了交联的明确识别。我们的创新分析概念以牛血清白蛋白（BSA）、野生型肿瘤抑制因子 p53、一种固有无序的蛋白质和视网膜鸟苷酸环化酶激活蛋白-2（GCAP-2）为例进行了说明和应用。
