Quantitative Hydrogen/Deuterium Exchange Mass Spectrometry.

Abstract

This Account describes considerations for the data generation, data analysis, and data interpretation of a hydrogen/deuterium exchange-mass spectrometry (HDX-MS) experiment to have a quantitative argument. Although HDX-MS has gained its popularity as a biophysical tool, the argument from its data often remains qualitative. To generate HDX-MS data that are more suitable for a quantitative argument, the sequence coverage and sequence resolution should be optimized during the feasibility stage, and the time window coverage and time window resolution should be improved during the HDX stage. To extract biophysically meaningful values for a certain perturbation from medium-resolution HDX-MS data, there are two major ways: (i) estimating the area between the two deuterium buildup curves using centroid values with and without the perturbation when plotted against log time scale and (ii) dissecting into multiple single-exponential curves using the isotope envelopes. To have more accurate arguments for an HDX-MS perturbation study, (i) false negatives due to sequence coverage, (ii) false negatives due to time window coverage, (iii) false positives due to sequence resolution, and (iv) false positives due to allosteric effects should be carefully examined.