Department of Physiology and Biophysics, University of California, Irvine, California, USA.
Department of Chemistry, University of California, Irvine, California, USA.
Mol Cell Proteomics. 2021;20:100084. doi: 10.1016/j.mcpro.2021.100084. Epub 2021 Apr 27.
Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions nontargetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions because of nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MS. The MS-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.
交联质谱（XL-MS）是研究蛋白质-蛋白质相互作用和阐明蛋白质复合物结构的有力工具。虽然残基特异性 XL-MS 研究非常成功，但特定化学方法无法靶向的相互作用区域仍然难以研究。光化学由于其非特异性反应性显示出很大的潜力来捕获这些区域，但光交联产物的低产率和高复杂性阻碍了它们的鉴定，限制了当前研究主要集中在单个蛋白质上。在这里，我们描述了三种新型 MS 可裂解的杂双功能交联剂的开发，即 SDASO（琥珀酰亚胺基二氮杂环丁烷亚砜），用于通过 MS 快速准确地鉴定光交联肽。基于 MS 的工作流程允许对酵母 26S 蛋白酶体进行 SDASO XL-MS 分析，首次证明了对大型蛋白质复合物进行光交联的可行性。比较分析表明，SDASO 交联是稳健的，并且可以捕获与残基特异性试剂互补的相互作用，为未来在复杂 XL-MS 研究中应用光交联提供了基础。
