Estimating Constraints for Protection Factors from HDX-MS Data

Abstract Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry (MS) is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time dependent change in mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens but determining the latter from the former is generally not possible. Here we show that, if time-resolved measurements are available for a number of overlapping peptides that cover the whole sequence, rate constants for each amide hydrogen exchange (or equivalently, their protection factors) may be extracted, the uniqueness of the solutions obtained depending on the degree of peptide overlap. However, in most cases, the solution is not unique, and multiple alternatives must be considered. We provide a statistical method which clusters the solutions to further reduce their number. Such analysis always provides meaningful constraints on protection factors, and can be used in situations where obtaining more refined experimental data is impractical. It also provides a systematic way to improve data collection strategies in order to obtain unambiguous information at single residue level, e.g. for assessing protein structure predictions at atomistic level.