doi: 10.1021/ac060693n.
Supercritical fluid chromatography reduction of hydrogen/deuterium back exchange in solution-phase hydrogen/deuterium exchange with mass spectrometric analysis
- PMID: 17007536
- DOI: 10.1021/ac060693n
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Supercritical fluid chromatography reduction of hydrogen/deuterium back exchange in solution-phase hydrogen/deuterium exchange with mass spectrometric analysis
Anal Chem.
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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.
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