Dollar Ashlyn N, Webb Ian K
Department of Chemistry and Chemical Biology, Indiana University Indianapolis, Indianapolis, Indiana 46202, United States.
Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States.
Anal Chem. 2025 Apr 22;97(15):8213-8219. doi: 10.1021/acs.analchem.4c03978. Epub 2025 Apr 10.
Cross-linking and covalent labeling are common tools for protein structural elucidation by mass spectrometry. However, despite the importance of electrostatic interactions in proteins, there are not many tools for probing both acids and amines. Therefore, we introduce novel solution-phase amine-to-acid cross-linkers and single reagent amine and acid covalent labels utilizing carbodiimide chemistry. Cross-linking and labeling sites were determined using top-down and enzymatic digestion approaches. Ubiquitin was chosen as a model protein for isotopically labeled C-N-glycine cross-linking/covalent labeling and glycyl-l-proline cross-linking/covalent labeling with top-down mass spectrometry as a proof of concept. Alpha synuclein, an intrinsically disordered protein that plays a role in multiple neurological disorders, was also cross-linked/covalent labeled with these methods/reagents. We expect that these structural characterization techniques will play a role in gaining insight into functionally and pathologically important structural tendencies of disordered proteins.
交联和共价标记是通过质谱法阐明蛋白质结构的常用工具。然而，尽管静电相互作用在蛋白质中很重要，但用于探测酸和胺的工具并不多。因此，我们利用碳二亚胺化学方法引入了新型的溶液相胺到酸交联剂以及单一试剂胺和酸共价标记物。使用自上而下和酶切消化方法确定交联和标记位点。选择泛素作为模型蛋白，用于同位素标记的C-N-甘氨酸交联/共价标记以及甘氨酰-L-脯氨酸交联/共价标记，并采用自上而下的质谱法作为概念验证。α-突触核蛋白是一种在多种神经疾病中起作用的内在无序蛋白，也使用这些方法/试剂进行了交联/共价标记。我们期望这些结构表征技术将有助于深入了解无序蛋白在功能和病理方面重要的结构趋势。
