Real-time hydrogen/deuterium exchange kinetics via supercharged electrospray ionization tandem mass spectrometry.
Real-time hydrogen/deuterium exchange kinetics via supercharged electrospray ionization tandem mass spectrometry.
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DOI：
10.1021/ac101957x
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发表时间：
2010-11-01
影响因子：
7.4
通讯作者：
Williams, Evan R.
中科院分区：
文献类型：
--
作者：
Sterling, Harry J.;Williams, Evan R.
采用自上而下的电子转移解离（ETD）串联质谱（MS）方法，获得了牛泛素在含1%电喷雾离子化（ESI）“增压”试剂间硝基苯甲醇（m-NBA）的乙酸铵溶液中的酰胺氢/氘交换（HDX）速率常数。增压试剂通过在ESI液滴中引起快速蛋白质变性来取代HDX实验的常规MS方法中的酸和温度“淬灭”步骤。用m-NBA产生的更高电荷态离子更多地展开，如通过离子迁移率测量的，并且导致更高的片段化效率和更高的ETD序列覆盖率。单个氨基酸的分辨率获得了44个72个可交换的酰胺位点，并总结动力学数据获得的蛋白质的区域，相邻的碎片离子没有观察到，导致在1.3个残基的整体空间分辨率。将这些结果与来自NMR的先前值进行比较表明，增压试剂不会对初始ESI溶液中的蛋白质造成显著的结构变化，并且扰乱或反交换是最小的。这种自上而下的HDX-MS新方法能够在生理条件下进行实时动力学数据测量，类似于使用NMR获得的数据，具有可比的空间分辨率和显着更好的灵敏度。
Amide hydrogen/deuterium exchange (HDX) rate constants of bovine ubiquitin in an ammonium acetate solution containing 1% of the electrospray ionization (ESI) “supercharging” reagent m-nitrobenzyl alcohol (m-NBA) were obtained using top-down, electron transfer dissociation (ETD) tandem mass spectrometry (MS). The supercharging reagent replaces the acid and temperature “quench” step in the conventional MS approach to HDX experiments by causing rapid protein denaturation to occur in the ESI droplet. The higher charge state ions that are produced with m-NBA are more unfolded, as measured by ion mobility, and result in higher fragmentation efficiency and higher sequence coverage with ETD. Single amino acid resolution was obtained for 44 of 72 exchangeable amide sites, and summed kinetic data were obtained for regions of the protein where adjacent fragment ions were not observed, resulting in an overall spatial resolution of 1.3 residues. Comparison of these results with previous values from NMR indicates that the supercharging reagent does not cause significant structural changes to the protein in the initial ESI solution, and that scrambling or back-exchange is minimal. This new method for top-down HDX-MS enables real-time kinetic data measurements under physiological conditions, similar to those obtained using NMR, with comparable spatial resolution and significantly better sensitivity.
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DOI：
10.1016/j.jasms.2006.06.006
发表时间：
2006-11-01
影响因子：
3.2
作者：
Englander, S. Walter
通讯作者：
Englander, S. Walter
影响因子：
15
作者：
Breuker, K;Oh, HB;McLafferty, FW
通讯作者：
McLafferty, FW
影响因子：
7.4
作者：
Charlebois, JP;Patrie, SM;Kelleher, NL
通讯作者：
Kelleher, NL
影响因子：
7.4
作者：
Kaltashov, Igor A.;Bobst, Cedric E.;Abzalimov, Rinat R.
通讯作者：
Abzalimov, Rinat R.
DOI：
10.1016/0003-9861(57)90478-2
发表时间：
1957-01-01
期刊：
ARCH BIOCHEM AND BIOPHYS
影响因子：
--
作者：
BERGER, ARIEH;LINDERSTROM-LANG, K.
通讯作者：
LINDERSTROM-LANG, K.