Expansion of time window for mass spectrometric measurement of amide hydrogen/deuterium exchange reactions
Expansion of time window for mass spectrometric measurement of amide hydrogen/deuterium exchange reactions
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DOI：
10.1002/rcm.4814
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发表时间：
2010-12-01
影响因子：
2
通讯作者：
Hamuro, Yoshitomo
中科院分区：
文献类型：
--
作者：
Coales, Stephen J.;E, Sook Yen;Hamuro, Yoshitomo
主链酰胺氢交换率可用于描述蛋白质的动态特性。天然蛋白质中的酰胺氢交换率可能从几毫秒 (ms) 到几年不等。理想情况下，分析物蛋白质的所有酰胺氢的比率可以单独确定。为了实现这一目标，除了高序列覆盖率和高序列分辨率之外，更宽时间窗口的监测也至关重要。在过去的十年中，氢/氘交换质谱方法取得了重大改进，以获得更好的序列覆盖率和更高的序列分辨率。另一方面，很少有人努力扩大实验时间窗口以准确确定酰胺氢的交换率。许多快速交换的酰胺氢在典型的短交换时间点（10-30秒）完成之前完全交换，并且许多慢速交换的酰胺氢在典型的长交换时间点（1-3小时）之前不开始交换。这里，当转换成标准交换条件（pH 7和23摄氏度）时，利用各种实验条件以及骤冷装置来监测细胞色素c酰胺氢交换行为超过八个数量级（0.0044-1 000 000 s）。版权所有 (c) 2010 John Wiley & Sons, Ltd.
Backbone amide hydrogen exchange rates can be used to describe the dynamic properties of a protein. Amide hydrogen exchange rates in a native protein may vary from milliseconds (ms) to several years. Ideally, the rates of all amide hydrogens of the analyte protein can be determined individually. To achieve this goal, monitoring of a wider time window is critical, in addition to high sequence coverage and high sequence resolution. Significant improvements have been made to hydrogen/deuterium exchange mass spectrometry methods in the past decade for better sequence coverage and higher sequence resolution. On the other hand, little effort has been made to expand the experimental time window to accurately determine exchange rates of amide hydrogens. Many fast exchanging amide hydrogens are completely exchanged before completion of a typical short exchange time point (10-30 s) and many slow exchanging amide hydrogens do not start exchanging before a typical long exchanging time point (1-3 h). Here various experimental conditions, as well as a quenched-flow apparatus, are utilized to monitor cytochrome c amide hydrogen exchange behaviors over more than eight orders of magnitude (0.0044-1 000 000 s), when converted into the standard exchange condition (pH 7 and 23 degrees C). Copyright (c) 2010 John Wiley & Sons, Ltd.