Cross-linking mass spectrometry (XL-MS) has become a powerful structural tool for defining protein-protein interactions (PPIs) and elucidating architectures of large protein assemblies. To advance XL-MS studies, we have previously developed a series of sulfoxide-containing MS-cleavable cross-linkers to facilitate the detection and identification of cross-linked peptides using multistage mass spectrometry (MSn). While current sulfoxide-based cross-linkers are effective for in vivo and in vitro XL-MS studies at the systems-level, new reagents are still needed to help expand PPI coverage. To this end, we have designed and synthesized six variable-length derivatives of disuccinimidyl sulfoxide (DSSO) to better understand the effects of spacer arm modulation on MS-cleavability, fragmentation characteristics, and MS identification of cross-linked peptides. In addition, the impact on cross-linking reactivity was evaluated. Moreover, alternative MS2-based workflows were explored to determine their feasibility for analyzing new sulfoxide-containing cross-linked products. Based on the results of synthetic peptides and a model protein, we have further demonstrated the robustness and predictability of sulfoxide chemistry in designing MS-cleavable cross-linkers. Importantly, we have identified a unique asymmetric design that exhibits preferential fragmentation of cross-links over peptide backbones, a desired feature for MSn analysis. This work has established a solid foundation for further development of sulfoxide-containing MS-cleavable cross-linkers with new functionalities.

中文翻译：

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探索用于设计不对称亚砜 MS 可裂解交联剂的间隔臂结构。

交联质谱 (XL-MS) 已成为定义蛋白质-蛋白质相互作用 (PPI) 和阐明大型蛋白质组装体结构的强大结构工具。为了推进 XL-MS 研究，我们之前开发了一系列含亚砜 MS 可裂解的交联剂，以促进使用多级质谱 (MSn) 检测和鉴定交联肽。虽然当前基于亚砜的交联剂对于系统水平的体内和体外 XL-MS 研究有效，但仍然需要新的试剂来帮助扩大 PPI 覆盖范围。为此，我们设计并合成了六种可变长度的二琥珀酰亚胺亚砜（DSSO）衍生物，以更好地了解间隔臂调节对交联肽的MS裂解性、断裂特征和MS鉴定的影响。此外，还评估了对交联反应性的影响。此外，还探索了基于 MS2 的替代工作流程，以确定其分析新的含亚砜交联产品的可行性。基于合成肽和模型蛋白的结果，我们进一步证明了亚砜化学在设计 MS 可裂解交联剂中的稳健性和可预测性。重要的是，我们已经确定了一种独特的不对称设计，与肽主链相比，它表现出交联的优先断裂，这是 MSn 分析所需的特征。这项工作为进一步开发具有新功能的含亚砜MS可裂解交联剂奠定了坚实的基础。