Fast Reversed-Phase Liquid Chromatography to Reduce Back Exchange and Increase Throughput in H/D Exchange Monitored by FT-ICR Mass Spectrometry

Hui Min Zhang, George M. Bou-Assaf, Mark Emmett, Alan G. Marshall

Research output: Contribution to journalArticle

53 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)520-524
Number of pages5
JournalJournal of the American Society for Mass Spectrometry
Volume20
Issue number3
DOIs
StatePublished - Mar 2009
Externally publishedYes

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Liquid chromatography
Reverse-Phase Chromatography
Mass spectrometry
Hydrogen
Mass Spectrometry
Ion exchange
Deuterium
Throughput
Protein Conformation
Peptide Fragments
Proteins
Amides
Liquid Chromatography
Digestion
Amino Acid Sequence
High Pressure Liquid Chromatography
Ligands
Temperature
Phase separation
Conformations

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

Cite this

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; Bou-Assaf, George M.; Emmett, Mark; Marshall, Alan G.

In: Journal of the American Society for Mass Spectrometry, Vol. 20, No. 3, 03.2009, p. 520-524.

Research output: Contribution to journalArticle

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