A Novel MS-Cleavable Azo Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS)
A Novel MS-Cleavable Azo Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS)
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DOI：
10.1007/s13361-017-1744-6
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发表时间：
2017-10-01
影响因子：
3.2
通讯作者：
Sinz, Andrea
中科院分区：
文献类型：
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作者：
Iacobucci, Claudio;Hage, Christoph;Sinz, Andrea
化学交联/质谱 (MS) 方法是结构蛋白质组学中不断发展的研究领域，可以深入了解蛋白质构象。它依赖于在交联氨基酸侧链之间创建距离限制，可进一步用于衍生蛋白质结构。目前，设计新颖的交联原理的最紧迫任务是对所创建的交联产物进行明确且自动的分配。在这里，我们介绍了同双功能、胺反应性和水溶性交联剂偶氮二亚胺酯（ABI）作为一类新型交联剂的原型。 ABI-linker 拥有创新的模块化支架，结合了碰撞激活不稳定性和开壳化学的优点。这种 MS 可裂解的交联剂可以在正电离模式下通过自由基引发肽测序 (FRIPS) 进行有效操作。我们的原理验证研究挑战了赖氨酸、亮氨酸和异亮氨酸这三种氨基酸以及模型肽胸腺五肽的 ABI 连接体的气相行为。异构氨基酸亮氨酸和异亮氨酸可以通过其特征侧链片段来区分。碰撞激活实验是通过两台 Orbitrap 质谱仪上的正电喷雾电离 (ESI) 进行的。对 MS/MS 和 MS3 实验中 ABI 介导的奇数电子产物离子的形成进行了评估，并与之前描述的偶氮基交联剂进行了比较。所有交联产物均可通过 MeroX 软件进行自动分析，凸显了 ABI 连接器在结构蛋白质组学研究中的未来潜力。
The chemical cross-linking/mass spectrometry (MS) approach is a growing research field in structural proteomics that allows gaining insights into protein conformations. It relies on creating distance constraints between cross-linked amino acid side chains that can further be used to derive protein structures. Currently, the most urgent task for designing novel cross-linking principles is an unambiguous and automated assignment of the created cross-linked products. Here, we introduce the homobifunctional, amine-reactive, and water soluble cross-linker azobisimidoester (ABI) as a prototype of a novel class of cross-linkers. The ABI-linker possesses an innovative modular scaffold combining the benefits of collisional activation lability with open shell chemistry. This MS-cleavable cross-linker can be efficiently operated via free radical initiated peptide sequencing (FRIPS) in positive ionization mode. Our proof-of-principle study challenges the gas phase behavior of the ABI-linker for the three amino acids, lysine, leucine, and isoleucine, as well as the model peptide thymopentin. The isomeric amino acids leucine and isoleucine could be discriminated by their characteristic side chain fragments. Collisional activation experiments were conducted via positive electrospray ionization (ESI) on two Orbitrap mass spectrometers. The ABI-mediated formation of odd electron product ions in MS/MS and MS3 experiments was evaluated and compared with a previously described azo-based cross-linker. All cross-linked products were amenable to automated analysis by the MeroX software, underlining the future potential of the ABI-linker for structural proteomics studies.