Supercritical Fluid Chromatography Reduction of Hydrogen/Deuterium Back Exchange in Solution-Phase Hydrogen/Deuterium Exchange with Mass Spectrometric Analysis

摘要

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 deg C) HPLC was performed to help reduce back-exchange, but calculated back-exchange still averages approx30percent. 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.