Novel MS/MS-Cleavable Cross-Linkers: Synthesis, Evaluation of Fragmentation Behavior, and Application for Protein Structure Analysis

化学交联质谱(MS)已发展成为阐明蛋白质三维结构和绘制蛋白质界面的一种真正的替代策略。这里概述的联合研究资助提案继续了我们在推进交叉链接/MS方法方面卓有成效的合作，该方法以前一直由DFG资助。我们将继续充分探索碰撞诱导解离(CID)不稳定试剂的优越能力，这些试剂以前为生物相关蛋白质体系的三级和四级结构阐明而开发，我们的目标是通过进一步优化和调整反应性(改善的水溶解性：磺酸衍生物；C末端的反应性：联吡肼类似物；引入溴化版本的我们的尿素连接物，通过精确的离子质量测量和同位素模式评估进行选择性检测)来扩大CID裂解试剂的适用性。此外，我们将加紧努力，进一步完善我们为自动数据采集和解释而设置的定制软件工具和算法，因为生物信息学已被证明对大规模交联研究的成功至关重要(MS3实验中检测交联剂修饰的多肽的触发恒定中性损失(CNL))。此外，一类新的具有四氢嘧啶部分的CID可裂解交联剂被提出用于交联目的，最好通过RDA反应途径解离。我们将使用所有可用的串联MS方法来测试其CID特性，即CID、HCD(高能碰撞诱导解离)、电子转移解离(ETD)和电子俘获解离ECD以及不同碎裂技术的组合(ETciD和EThcD)。

Chemical cross-linking in combination with mass spectrometry (MS) has evolved into a real alternative strategy for elucidating threedimensional protein structures and for mapping protein interfaces. The joint research grant proposal outlined herein continues our fruitful collaboration in advancing the cross-linking/MS approach, which has previously been funded by the DFG. We will continue to fully explore the superior capabilities of the collision induced dissociation (CID)-labile reagents previously developed for tertiary and quaternary structure elucidation of biologically relevant protein systems and we aim to extend the applicability of the CID-cleavable reagents by further optimization and tuning of reactivity (improved water solubility: sulfonic acid derivatives; C-terminal reactivity: bishydrazide analogues; introduction of a brominated version of our urea-linker for selective detection via accurate ion mass measurements and isotopic pattern evaluation). Additionally, we will intensify our efforts to further refine our customized software tools and algorithms set in place for automated data acquisition and interpretation as bioinformatics has proven to be of vital importance for the success of large scale crosslinking studies (triggered constant neutral loss (CNL) for the detection of cross-linker modified peptides in MS3 experiments). Additionally, a new class of CID-cleavable cross-linkers with a tetrahydro-pyrimidine moiety is proposed for cross-linking purposes that should preferably dissociate via a retro-Diels-Alder (RDA) reaction pathway. All means of tandem MS methods that are available to us will be applied to test its CID characteristics, namely CID, HCD (higher-energy collisioninduced dissociation), electron transfer dissociation (ETD), and electron capture dissociation ECD as well as combinations of different fragmentation techniques (ETciD and EThcD).