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

新型 MS/MS 可裂解交联剂：合成、断裂行为评估以及在蛋白质结构分析中的应用

基本信息

批准号：
196601425
负责人：
Professor Dr. Mathias Schäfer
金额：
--
依托单位：
Institut für Organische Chemie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2011
资助国家：
德国
起止时间：
2010-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/196601425?language=en
关键词：
Novel MS Cleavable Cross Linkers

项目摘要

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).

化学交联与质谱（MS）相结合已经发展成为一种真正的替代策略，用于阐明三维蛋白质结构和绘制蛋白质界面。本文所述的联合研究资助提案继续了我们在推进交联/质谱方法方面卓有成效的合作，该方法此前已获得DFG的资助。我们将继续充分挖掘碰撞诱导解离（CID）不稳定试剂的优势能力，之前开发的用于生物相关蛋白质体系的三级和四级结构解析的试剂，我们的目标是通过进一步优化和调整反应性(改善水溶性：磺酸衍生物；c端反应性：双酰肼类似物；引入溴化版本的尿素连接剂，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)。此外，我们将加强努力，进一步完善我们定制的软件工具和算法，用于自动数据采集和解释，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（在MS3实验中检测交联剂修饰肽的触发恒定中性损失（CNL））。此外，提出了一类具有四氢嘧啶片段的新型cid可切割交联剂，用于交联目的，该交联剂最好通过反diols - alder （RDA）反应途径解离。我们将使用所有可用的串联质谱方法来测试其CID特性，即CID， HCD（高能碰撞诱导解离），电子转移解离（ETD），电子捕获解离ECD以及不同碎片技术的组合（ETciD和EThcD）。