Polar aprotic modifiers for chromatographic separation and back-exchange reduction for protein hydrogen/deuterium exchange monitored by fourier transform ion cyclotron resonance mass spectrometry

Santosh G. Valeja, Mark R. Emmett, Alan G. Marshall

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Hydrogen/deuterium exchange monitored by mass spectrometry is an important non-perturbing tool to study protein structure and protein-protein interactions. However, water in the reversedphase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast high-performance liquid chromatography (HPLC) and supercritical fluid chromatography have been shown to decrease back-exchange. Here, we show that replacement of up to 40% of the water in the LC mobile phase by the modifiers, dimethylformamide (DMF) and N-methylpyrrolidone (NMP) (i.e., polar organic modifiers that lack rapid exchanging hydrogens), significantly reduces back-exchange. On-line LC micro-ESI FT-ICR MS resolves overlapped proteolytic peptide isotopic distributions, allowing for quantitative determination of the extent of back-exchange. The DMF modified solvent composition also improves chromatographic separation while reducing back-exchange relative to conventional solvent.

Original languageEnglish (US)
Pages (from-to)699-707
Number of pages9
JournalJournal of the American Society for Mass Spectrometry
Volume23
Issue number4
DOIs
StatePublished - Apr 1 2012
Externally publishedYes

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Keywords

  • FT-ICR
  • FTMS
  • Luteinizing hormone releasing hormone (LHRH)
  • Myoglobin

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

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