Zhang Yanxin, Yan Qibo, Li Ming, Liu Hailong, Liao Weiwei, Wang Yingwu, Duan Haifeng, Wei Zhonglin, Lin Yingjie
Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130012, Jilin, China.
Division of Chemical Metrology & Analytical Science, National Institute of Metrology, Beijing 100029, China.
Anal Chem. 2025 Jul 22;97(28):15190-15198. doi: 10.1021/acs.analchem.5c01695. Epub 2025 Jul 11.
Chemical cross-linking mass spectrometry (XL-MS) is crucial for probing protein structure and protein-protein interactions. Currently, lysine-reactive cross-linkers have been widely developed and applied. In this study, two urazole-derived MS-cleavable cross-linkers were designed and synthesized, enabling the accurate identification of cross-linked peptides by MS. A homobifunctional cross-linker (sulfoxide, bis-urazolyl, SBT) was designed to target tyrosine residues through the electrochemical click reaction. Another heterobifunctional cross-linker (sulfoxide, succinimidyl, urazolyl, SCT) was synthesized to target lysine and tyrosine residues. The reaction efficiencies of cross-linkers were systematically evaluated by using them to react with peptides, proteins, and protein complexes, respectively. Additionally, five structures of BSA, derived from AlphaFold 3, were analyzed by comparing them with its crystal structure in the protein database. The results demonstrate that this type of urazole-derived cross-linker is a valuable addition to the existing cross-linker library and enhances the recognition coverage of XL-MS in the structural analysis of proteins.
化学交联质谱法（XL-MS）对于探究蛋白质结构和蛋白质-蛋白质相互作用至关重要。目前，赖氨酸反应性交联剂已得到广泛开发和应用。在本研究中，设计并合成了两种基于脲唑的可质谱裂解交联剂，能够通过质谱准确鉴定交联肽段。设计了一种同双功能交联剂（亚砜、双脲唑基，SBT），通过电化学点击反应靶向酪氨酸残基。合成了另一种异双功能交联剂（亚砜、琥珀酰亚胺基、脲唑基，SCT），以靶向赖氨酸和酪氨酸残基。分别使用这些交联剂与肽段、蛋白质和蛋白质复合物反应，系统评估了交联剂的反应效率。此外，通过将来自AlphaFold 3的牛血清白蛋白的五种结构与其在蛋白质数据库中的晶体结构进行比较来进行分析。结果表明，这种基于脲唑的交联剂是现有交联剂库中的宝贵补充，并提高了XL-MS在蛋白质结构分析中的识别覆盖率。
