Supercritical fluid chromatography reduction of hydrogen/deuterium back exchange in solution-phase hydrogen/deuterium exchange with mass spectrometric analysis.
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.
Emmett
Mark R
MR
Kazazic
Sasa
S
Marshall
Alan G
AG
Chen
Wei
W
Shi
Stone D-H
SD
Bolaños
Ben
B
Greig
Michael J
MJ
D000595:Amino Acid Sequence; D025924:Chromatography, Supercritical Fluid / Q000379:methods*; D003903:Deuterium / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry
D009211:Myoglobin; D006859:Hydrogen; D003903:Deuterium