Recommendations for the Propagation of Uncertainty in Hydrogen Exchange-Mass Spectrometric Measurements.

Abstract

Hydrogen exchange-mass spectrometry (HX-MS) is used widely to characterize higher-order protein structure and to locate changes in protein structure and dynamics that accompany, for example, ligand binding and protein-protein interactions. Quantitative differences in the amount of hydrogen exchange between two states (i.e., differential HX) are taken as evidence of significant differences in higher-order structure or dynamics. The quantitative measures range from simple mass differences at one HX labeling time to differences averaged across an HX time course with correction for deuterium recovery. This work applies the principles of uncertainty propagation to differential HX measurements to facilitate the identification of significant differences. Furthermore, it is shown that pooled estimates of experimental uncertainty result in a lower false positive rate than estimates of uncertainty based on individual standard deviations.