Fast reversed-phase liquid chromatography to reduce back exchange and increase throughput in H/D exchange monitored by FT-ICR mass spectrometry.
Fast reversed-phase liquid chromatography to reduce back exchange and increase throughput in H/D exchange monitored by FT-ICR mass spectrometry.
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DOI：
10.1016/j.jasms.2008.11.010
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发表时间：
2009-03
影响因子：
3.2
通讯作者：
Marshall AG
中科院分区：
文献类型：
--
作者：
Zhang HM;Bou-Assaf GM;Emmett MR;Marshall AG
在溶液相氢/氘交换（HDX）中，在交换被淬灭后，使H对D的反交换水平最小化是至关重要的，以准确地分配蛋白质构象和蛋白质-蛋白质或蛋白质-配体相互作用。反相HPLC在低pH和低温下进行，以脱盐和分离蛋白水解片段。然而，由于在移动的相中长时间暴露于H2O，反交换平均约30%。在本文中，我们首先表明，有没有显着的骨架酰胺氢回交换过程中淬火和消化，骨架交换主要发生在随后的LC分离。然后，我们表明，快速的反相分离减少了至少25%的HDX交换回，由于显着减少的保留时间的肽片段在柱上。同时考察了保留时间对反交换的影响。快速分离与14.5T下的高分辨率FT-ICR MS相结合，提供了高氨基酸序列覆盖率，高样品通量以及高重现性和可靠性。
In solution-phase hydrogen/deuterium exchange (HDX), it is essential to minimize the back exchange level of H for D after the exchange has been quenched, to accurately assign protein conformation and protein-protein or protein-ligand interactions. Reversed-phase HPLC is conducted at low pH and low temperature to desalt and separate proteolytic fragments. However, back exchange averages ∼30% due to long exposure to H2O in the mobile phase. In this paper, we first show that there is no significant backbone amide hydrogen back exchange during quenching and digestion; backbone exchange occurs primarily during subsequent LC separation. We then show that a rapid reversed-phase separation reduces back exchange for HDX by at least 25%, due to dramatically reduced retention time of the peptide fragments on the column. The influence of retention time on back exchange was also evaluated. The rapid separation coupled with high resolution FT-ICR MS at 14.5T provides high amino acid sequence coverage, high sample throughput, and high reproducibility and reliability.
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影响因子：
7.4
作者：
Zhang, Hui-Min;Kazazic, Sasa;Schaub, Tanner M.;Tipton, Jeremiah D.;Emmett, Mark R.;Marshall, Alan G.
通讯作者：
Marshall, Alan G.
影响因子：
4.1
作者：
Lam, TT;Lanman, JK;Prevelige, PE
通讯作者：
Prevelige, PE
DOI：
10.1002/prot.340170110
发表时间：
1993-09-01
期刊：
PROTEINS-STRUCTURE FUNCTION AND GENETICS
影响因子：
--
作者：
BAI, YW;MILNE, JS;ENGLANDER, SW
通讯作者：
ENGLANDER, SW
影响因子：
7.4
作者：
Emmett, Mark R.;Kazazic, Sasa;Greig, Michael J.
通讯作者：
Greig, Michael J.
影响因子：
7.4
作者：
Engen, JR;Smith, DL
通讯作者：
Smith, DL