Zhang Hui-Min, Bou-Assaf George M, Emmett Mark R, Marshall Alan G
National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32310-4005, USA.
J Am Soc Mass Spectrom. 2009 Mar;20(3):520-4. doi: 10.1016/j.jasms.2008.11.010. Epub 2008 Nov 21.
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 roughly 30% because of the long exposure to H(2)O in the mobile phase. In this report, we first show that there is no significant backbone amide hydrogen back exchange during quench and digestion; backbone exchange occurs primarily during subsequent liquid chromatography separation. We then show that a rapid reversed-phase separation reduces back exchange for HDX by at least 25%, resulting from the 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 14.5 T provides high amino acid sequence coverage, high sample throughput, and high reproducibility and reliability.
在溶液相氢/氘交换（HDX）中，在交换淬灭后将H重新交换为D的水平降至最低至关重要，以便准确确定蛋白质构象以及蛋白质-蛋白质或蛋白质-配体相互作用。反相高效液相色谱在低pH和低温下进行，以脱盐并分离蛋白水解片段。然而，由于在流动相中长时间暴露于H₂O，反向交换平均约为30%。在本报告中，我们首先表明在淬灭和消化过程中没有显著的主链酰胺氢反向交换；主链交换主要发生在随后的液相色谱分离过程中。然后我们表明，快速反相分离可将HDX的反向交换降低至少25%，这是由于肽片段在柱上的保留时间显著缩短所致。还评估了保留时间对反向交换的影响。在14.5 T下的快速分离与高分辨率傅里叶变换离子回旋共振质谱（FT-ICR MS）相结合，可提供高氨基酸序列覆盖率、高样品通量以及高重现性和可靠性。
