Protein-protein interaction is one of the key regulatory mechanisms for controlling protein function in various cellular processes. Chemical cross-linking coupled with mass spectrometry has proven to be a powerful method not only for mapping protein-protein interactions of all natures, including weak and transient ones, but also for determining their interaction interfaces. One critical challenge remaining in this approach is how to effectively isolate and identify cross-linked products from a complex peptide mixture. In this work, we have developed a novel strategy using conjugation chemistry for selective enrichment of cross-linked products. An azide-tagged cross-linker along with two biotinylated conjugation reagents were designed and synthesized. Cross-linking of model peptides and cytochrome c as well as enrichment of the resulting cross-linked peptides has been assessed. Selective conjugation of azide-tagged cross-linked peptides has been demonstrated using two strategies: copper catalyzed cycloaddition and Staudinger ligation. While both methods are effective, Staudinger ligation is better suited for enriching the cross-linked peptides since there are fewer issues with sample handling. LC MSⁿ analysis coupled with database searching using the Protein Prospector software package allowed identification of 58 cytochrome c cross-linked peptides after enrichment and affinity purification. The new enrichment strategy developed in this work provides useful tools for facilitating identification of cross-linked peptides in a peptide mixture by MS, thus presenting a step forward in future studies of protein-protein interactions of protein complexes by cross-linking and mass spectrometry.

蛋白质-蛋白质相互作用是控制各种细胞过程中蛋白质功能的关键调节机制之一。化学交联与质谱联用已被证明是一种强大的方法，不仅用于绘制所有性质的蛋白质 - 蛋白质相互作用，包括弱和瞬态相互作用，而且还用于确定它们的相互作用界面。这种方法剩下的一个关键挑战是如何有效地从复杂的肽混合物中分离和鉴定交联产物。在这项工作中，我们开发了一种使用共轭化学选择性富集交联产物的新策略。设计并合成了叠氮化物标记的交联剂和两种生物素化偶联试剂。模型肽和细胞色素c 的交联以及对所得交联肽的富集进行了评估。已经使用两种策略证明了叠氮化物标记的交联肽的选择性缀合：铜催化环加成和施陶丁格连接。虽然这两种方法都有效，但 Staudinger 连接更适合富集交联肽，因为样品处理问题较少。LC MS ⁿ分析结合使用 Protein Prospector 软件包进行数据库搜索，可识别 58 种细胞色素c富集和亲和纯化后的交联肽。这项工作中开发的新富集策略为促进通过 MS 鉴定肽混合物中的交联肽提供了有用的工具，从而在未来通过交联和质谱法研究蛋白质复合物的蛋白质 - 蛋白质相互作用方面向前迈进了一步。