Efficient Isothiocyanate Modification of Peptides Facilitates Structural Analysis by Radical-Directed Dissociation
Efficient Isothiocyanate Modification of Peptides Facilitates Structural Analysis by Radical-Directed Dissociation
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DOI：
10.1021/jasms.1c00237
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发表时间：
2022-08-03
影响因子：
3.2
通讯作者：
Julian, Ryan R.
中科院分区：
文献类型：
--
作者：
Lambeth, Tyler R.;Julian, Ryan R.
自由基定向解离（RDD）是质谱实验中肽段结构表征的一种有效技术。在分析之前，通常必须附加自由基前体以促进自由基的产生。为了探索可以在单个步骤中容易地连接的自由基前体的使用，我们使用与异硫氰酸碘苯酯的“点击”反应来修饰肽。与胺官能团的偶联以高产率进行，产生稳定的碘苯基硫脲修饰的肽。光解产率记录在266和213 nm的2-，3-和4-碘异构体的改性剂，并发现在几乎所有情况下是最高的4-碘异构体。碰撞活化后的改性肽片段显示有利的标签损失，和电子结构计算被用来评估一个潜在的机制，涉及氢转移内的硫脲基团。RDD数据的检查显示，4-碘苯甲酸，4-碘苯基硫脲，3-碘酪氨酸产生类似的碎片模式为一个给定的肽，虽然片段丰度的差异。异硫氰酸碘苯酯标记结合RDD可用于区分肽内的异构氨基酸，这应有助于简化复杂生物样品中存在的异构体的评价。
Radical-directed dissociation (RDD) is a powerful technique for structural characterization of peptides in mass spectrometry experiments. Prior to analysis, a radical precursor must typically be appended to facilitate generation of a free radical. To explore the use of a radical precursor that can be easily attached in a single step, we modified peptides using a "click" reaction with iodophenyl isothiocyanate. Coupling with amine functional groups proceeds with high yields, producing stable iodophenylthiourea-modified peptides. Photodissociation yields were recorded at 266 and 213 nm for the 2-, 3-, and 4-iodo isomers of the modifier and found to be highest for the 4-iodo isomer in nearly all cases. Fragmentation of the modified peptides following collisional activation revealed favorable losses of the tag, and electronic structure calculations were used to evaluate a potential mechanism involving hydrogen transfer within the thiourea group. Examination of RDD data revealed that 4-iodobenzoic acid, 4-iodophenylthiourea, and 3-iodotyrosine yield similar fragmentation patterns for a given peptide, although differences in fragment abundance are noted. Iodophenyl isothiocyanate labeling in combination with RDD can be used to differentiate isomeric amino acids within peptides, which should facilitate simplified evaluation of isomers present in complex biological samples.