Initial Protein Unfolding Events in Ubiquitin, Cytochrome c and Myoglobin Are Revealed with the Use of 213 nm UVPD Coupled to IM-MS.
Initial Protein Unfolding Events in Ubiquitin, Cytochrome c and Myoglobin Are Revealed with the Use of 213 nm UVPD Coupled to IM-MS.
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DOI：
10.1007/s13361-018-1992-0
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发表时间：
2019-01
影响因子：
3.2
通讯作者：
Barran PE
中科院分区：
文献类型：
--
作者：
Theisen A;Black R;Corinti D;Brown JM;Bellina B;Barran PE
蛋白质解折叠的初始阶段可以反映整个折叠的稳定性，也可以揭示蛋白质的哪些部分可以被扰动，而不会重组其余部分。在这项工作中，我们耦合UVPD与活化离子迁移率质谱测量如何三个模型蛋白质开始展开。泛素，细胞色素c和肌红蛋白离子产生通过nESI从盐溶液进行UV照射前迁移率分离;实验进行了一系列的源条件，改变的前体离子的构象所示的漂移时间曲线。对于所有这三种蛋白质，紧凑的结构导致更少的碎片比更扩展的结构，出现在渐进的源内激活。裂解位点被发现不同的构象合奏，例如，细胞色素c [M +7 H]7+的主要电荷状态，裂解在Phe 10，Thr 19和Val 20只观察到在激活条件下，而在Ala 43裂解显着增强。将光裂解片段映射到晶体结构上提供了对蛋白质展开过程中发生的局部结构变化的深入了解，这与漂移时间曲线中观察到的全局重组相结合。本文的在线版本（10.1007/s13361-018-1992-0）包含补充材料，可供授权用户使用。
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 The online version of this article (10.1007/s13361-018-1992-0) contains supplementary material, which is available to authorized users.
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