Supercritical fluid chromatography reduction of hydrogen/deuterium back exchange in solution-phase hydrogen/deuterium exchange with mass spectrometric analysis

Anal Chem. 2006 Oct 1;78(19):7058-60. doi: 10.1021/ac060693n.

Abstract

The single biggest problem with solution-phase H/D exchange as a mass spectrometric probe of surface exposure in a protein (or protein complex) is back-exchange of H for D after the initial H/D exchange has been quenched. Back-exchange results in loss of pertinent data and also greatly hampers data analysis. Previously, very fast, cold (0-4 degrees C) HPLC was performed to help reduce back-exchange, but calculated back-exchange still averages approximately 30%. In this report, supercritical fluid chromatography replaces HPLC as the desalting/separation technique prior to mass analysis, providing a dramatic reduction in back-exchange compared to the fast, cold HPLC methods.

Publication types

  • Letter
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Chromatography, Supercritical Fluid / methods*
  • Deuterium / chemistry*
  • Hydrogen / chemistry*
  • Mass Spectrometry / methods*
  • Molecular Sequence Data
  • Myoglobin / chemistry

Substances

  • Myoglobin
  • Hydrogen
  • Deuterium