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Initial Protein Unfolding Events in Ubiquitin, Cytochrome c and Myoglobin Are Revealed with the Use of 213 nm UVPD Coupled to IM-MS.
Journal of the American Society for Mass Spectrometry ( IF 3.1 ) Pub Date : 2018-06-13 , DOI: 10.1007/s13361-018-1992-0
Alina Theisen 1 , Rachelle Black 1 , Davide Corinti 2 , Jeffery M Brown 3 , Bruno Bellina 1 , Perdita E Barran 1
Affiliation  

The initial stages of protein unfolding may reflect the stability of the entire fold and can also reveal which parts of a protein can be perturbed, without restructuring the rest. In this work, we couple UVPD with activated ion mobility mass spectrometry to measure how three model proteins start to unfold. Ubiquitin, cytochrome c and myoglobin ions produced via nESI from salty solutions are subjected to UV irradiation pre-mobility separation; experiments are conducted with a range of source conditions which alter the conformation of the precursor ion as shown by the drift time profiles. For all three proteins, the compact structures result in less fragmentation than more extended structures which emerge following progressive in-source activation. Cleavage sites are found to differ between conformational ensembles, for example, for the dominant charge state of cytochrome c [M + 7H]7+, cleavage at Phe10, Thr19 and Val20 was only observed in activating conditions whilst cleavage at Ala43 is dramatically enhanced. Mapping the photo-cleaved fragments onto crystallographic structures provides insight into the local structural changes that occur as protein unfolding progresses, which is coupled to global restructuring observed in the drift time profiles. Graphical Abstract.

中文翻译:

使用 213 nm UVPD 与 IM-MS 耦合揭示了泛素、细胞色素 c 和肌红蛋白中的初始蛋白质展开事件。

蛋白质展开的初始阶段可能反映了整个折叠的稳定性,也可以揭示蛋白质的哪些部分可以被扰动,而不需要重组其余部分。在这项工作中,我们将 UVPD 与活化离子迁移质谱法相结合,以测量三种模型蛋白如何开始展开。通过 nESI 从盐溶液中产生的泛素、细胞色素 c 和肌红蛋白离子进行紫外线照射前迁移分离;实验是在一系列源条件下进行的,这些条件会改变前体离子的构象,如漂移时间曲线所示。对于所有三种蛋白质,紧凑结构比在源内激活后出现的更多扩展结构产生更少的碎片。发现裂解位点在构象集合之间存在差异,例如,对于细胞色素 c [M + 7H]7+ 的主要电荷状态,仅在激活条件下观察到 Phe10、Thr19 和 Val20 处的切割,而 Ala43 处的切割则显着增强。将光切割的片段映射到晶体结构上,可以深入了解随着蛋白质展开过程发生的局部结构变化,这与漂移时间曲线中观察到的全局重组相结合。图形概要。
更新日期:2018-06-13
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