†Laboratory of Mass Spectrometry, Department of Chemistry, GIGA-R, University of Liege, Allée de la Chimie 3, B-4000 Liege, Belgium.
‡Commissariat à l'Energie Atomique, DSV/iBiTec-S/SIMOPRO, F91191 Gif-sur-Yvette, France.
Anal Chem. 2015;87(10):5240-6. doi: 10.1021/acs.analchem.5b00245. Epub 2015 May 7.
Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability toward enzymatic degradations and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e., the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electron-transfer dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides, resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion mobility, and (iii) to characterize the species using collision-induced dissociation (CID). Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH· radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process.
二硫键是一种常见的翻译后修饰（PTMs），通常存在于肽和蛋白质中。它们增加了肽和蛋白质对酶降解的稳定性，并为这些折叠蛋白质提供了结构和（因此）活性。二硫键模式（即半胱氨酸连接性）的表征对于实现感兴趣蛋白质的活性构象的全局图景至关重要。电子转移解离（ETD）是在气相中切割二硫键的一种有价值的工具，避免了溶液中的化学还原/烷基化。为了表征半胱氨酸配对，本工作提出了（i）通过 ETD 还原模型肽中的两个二硫键之一，导致环状结构的打开，（ii）通过离子淌度分离生成的物质，以及（iii）使用碰撞诱导解离（CID）来表征物质。该策略应用于几种肽的结果表明，连接性不同，行为也不同。所有肽都观察到 SH·自由基物质的损失，这证实了 ETD 过程中二硫键的断裂。
