Presentation of a homobifunctional azo-reagent for protein structure analysis by collision-induced dissociative chemical cross-linking: Proof-of-principle
Presentation of a homobifunctional azo-reagent for protein structure analysis by collision-induced dissociative chemical cross-linking: Proof-of-principle
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DOI：
10.1016/j.ijms.2013.04.012
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发表时间：
2013-11-15
影响因子：
1.8
通讯作者：
Schaefer, Mathias
中科院分区：
文献类型：
--
作者：
Falvo, Francesco;Fiebig, Lukas;Schaefer, Mathias
4，4 '-偶氮双[4-氰基戊酸]（对称自由基引发剂）转化为双-N-琥珀酰亚胺基活性酯（azoXL）并用于肽交联（XL）。azoXL-试剂与胺官能团反应，并在碰撞诱导解离（CID）时仅进行N-2的损失，导致形成两个自由基位点。（+）N末端通过4，4 '-偶氮二[4-氰基戊酸]相互连接的两种模型肽（MRFA和RKDVY）的质子化分子离子的ESI-MSn产物离子光谱显示特征性质量位移产物离子和中性损失（N-2 = 28 Da; 2-甲基丙烯腈= 67 Da），可能用于鉴定XL衍生肽。此外，CID断裂反应导致形成序列特异性碎片离子，从而允许一级结构解析。所呈现的结果证明了偶氮化合物作为CID不稳定XL试剂用于串联MS分析的潜力。通过MSn、精确离子质量测量和与烯丙基碘和二甲基二硫醚的离子-分子反应（IMR）研究了用azoXL试剂衍生的肽（MRFA、RKDVY和P物质）的裂解行为。(C)2013爱思唯尔有限公司版权所有。
4,4'-Azobis[4-cyanopentanoic] acid, a symmetrical free radical initiator, is transformed in the bis-N-succinimidyl-active ester (azoXL) and utilized for peptide cross-linking (XL). The azoXL-reagent reacts with amine functionalities and performs upon collision induced dissociation (CID) exclusively the loss of N-2 leading to the formation of two radical sites. (+)ESI-MSn product-ion spectra of the protonated molecular ion of two model peptides (MRFA and RKDVY) interconnected N-terminally by 4,4'-azobis[4-cyanopentanoic] acid show characteristically mass-shifted product ions and neutral losses (N-2 = 28 Da; 2-methylacrylonitrile = 67 Da), potentially useful for identification of XL derivatized peptides. Additionally, CID fragmentation reactions lead to the formation of sequence-specific fragment ions, allowing primary structure elucidation. The presented results demonstrate the potential of azo-compounds as CID-labile XL-reagents for tandem MS analysis. The fragmentation behaviour of peptides (MRFA, RKDVY and Substance P) derivatized with the azoXL reagent is studied by MSn, exact ion mass measurements and ion-molecule reactions (IMRs) with allyliodide and dimethyl disulfide. (C) 2013 Elsevier B.V. All rights reserved.