Disulfide bond cleavage in TEMPO-free radical initiated peptide sequencing mass spectrometry
Disulfide bond cleavage in TEMPO-free radical initiated peptide sequencing mass spectrometry
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DOI：
10.1002/jms.1955
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发表时间：
2011-08-01
影响因子：
2.3
通讯作者：
Oh, Han Bin
中科院分区：
文献类型：
--
作者：
Lee, Minhee;Lee, Younjin;Oh, Han Bin
利用串联质谱实验研究了具有分子内和分子间二硫键的o-TEMPO-Bz缀合肽的气相自由基引发肽测序（FRIPS）裂解行为。所研究的肽包括具有分子内环状二硫键的四种肽，即Bactenecin（RLCRIVIRVCR）、TGF-α（CHSGYVGVRC）、MCH（DFDMLRCMLGRVFRPCWQY）和肾上腺髓质素（16-31）（CRFGTCTVQKLAHQIY），以及具有分子间二硫键的两种肽。通过在初始碰撞活化时从o-TEMPO-Bz缀合的肽释放克里思自由基而产生的苄基自由基缀合的肽阳离子的碰撞活化产生了大量的肽骨架片段，其中S-S或C-S键容易裂解。观察到的肽骨架片段包括a-、c-、x-或a-型，这表明自由基驱动的肽断裂机制在TEMPO FRIPS质谱中起着重要作用。线性连接的二硫键肽的FRIPS应用进一步表明，S-S或C-S键被选择性地和优先地切割，随后是肽骨架解离。在FRIPS质谱图中，也发现了大量的奥什或巯基抑制素的丢失。在这些发现的基础上，FRIPS裂解的多肽与二硫键的途径提出。对于S-S键的断裂，苄基自由基夺取C（β）处的氢原子被认为是初始自由基夺取/转移反应。另一方面，在C（alpha）处的H-提取被认为导致C-S键断裂，这产生[离子+/- S]片段或奥什或oSSH的损失。版权所有（C）2011约翰威利父子有限公司
The gas-phase free radical initiated peptide sequencing (FRIPS) fragmentation behavior of o-TEMPO-Bz-conjugated peptides with an intro- and intermolecular disulfide bond was investigated using MS(n) tandem mass spectrometry experiments. Investigated peptides included four peptides with an intramolecular cyclic disulfide bond, Bactenecin (RLCRIVVIRVCR), TGF-alpha (CHSGYVGVRC), MCH (DFDMLRCMLGRVFRPCWQY) and Adrenomedullin (16-31) (CRFGTCTVQKLAHQIY), and two peptides with an intermolecular disulfide bond. Collisional activation of the benzyl radical conjugated peptide cation, which was generated through the release of a TEMPO radical from o-TEMPO-Bz-conjugated peptides upon initial collisional activation, produced a large number of peptide backbone fragments in which the S-S or C-S bond was readily cleaved. The observed peptide backbone fragments included a-, c-, x- or a-types, which indicates that the radical-driven peptide fragmentation mechanism plays an important role in TEMPO-FRIPS mass spectrometry. FRIPS application of the linearly linked disulfide peptides further showed that the S-S or C-S bond was selectively and preferentially cleaved, followed by peptide backbone dissociations. In the FRIPS mass spectra, the loss of oSH or oSSH was also abundantly found. On the basis of these findings, FRIPS fragmentation pathways for peptides with a disulfide bond are proposed. For the cleavage of the S-S bond, the abstraction of a hydrogen atom at C(beta) by the benzyl radical is proposed to be the initial radical abstraction/transfer reaction. On the other hand, H-abstraction at C(alpha), is suggested to lead to C-S bond cleavage, which yields [ion +/- S] fragments or the loss of oSH or oSSH. Copyright (C) 2011 John Wiley & Sons, Ltd.