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Fast reversed-phase liquid chromatography to reduce back exchange and increase throughput in H/D exchange monitored by FT-ICR mass spectrometry.


ABSTRACT: 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.

SUBMITTER: Zhang HM 

PROVIDER: S-EPMC2673454 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Fast reversed-phase liquid chromatography to reduce back exchange and increase throughput in H/D exchange monitored by FT-ICR mass spectrometry.

Zhang Hui-Min HM   Bou-Assaf George M GM   Emmett Mark R MR   Marshall Alan G AG  

Journal of the American Society for Mass Spectrometry 20081121 3


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  ...[more]

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