Journal
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Volume 20, Issue 3, Pages 520-524Publisher
SPRINGER
DOI: 10.1016/j.jasms.2008.11.010
Keywords
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Funding
- NTH [TROT GM-78359]
- NSF Division of Materials Research [DMR-0654118]
- State of Florida
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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 H2O 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. (J Am Soc Mass Spectrom 2009, 20, 520-524) (C) 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry
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