A Residue-Resolved Bayesian Approach to Quantitative Interpretation of Hydrogen–Deuterium Exchange from Mass Spectrometry: Application to Characterizing Protein–Ligand Interactions

Abstract

Characterization of interactions between proteins and other molecules is crucial for understanding the mechanisms of action of biological systems and, thus, drug discovery. An increasingly useful approach to mapping these interactions is measurement of hydrogen/deuterium exchange (HDX) using mass spectrometry (HDX-MS), which measures the time-resolved deuterium incorporation of peptides obtained by enzymatic digestion of the protein. Comparison of exchange rates between apo- and ligand-bound conditions results in a mapping of the differential HDX (ΔHDX) of the ligand. Residue-level analysis of these data, however, must account for experimental error, sparseness and ambiguity due to overlapping peptides. Here, we propose a Bayesian method consisting of a forward model, noise model, prior probabilities, and a Monte Carlo sampling scheme. This method exploits a residue-resolved exponential rate model of HDX-MS data obtained from all peptides simultaneously, and explicitly models experimental error. The result is the best possible estimate of ΔHDX magnitude and significance for each residue given the data. We demonstrate the method by revealing richer structural interpretation of ΔHDX data on two nuclear receptors: vitamin D-receptor (VDR) and retinoic acid receptor gamma (RORγ). The method is implemented in HDX Workbench and as a standalone module of the open source Integrative Modeling Platform.

Assessing Exhaustiveness of Stochastic Sampling for Integrative Modeling of Macromolecular Structures

“…Finally, our statistical test applies to independent samples from a stochastic algorithm such as Monte Carlo sampling, whereas some other methods do not require the samples to be independent (36)(37)(38)(39)(40). Preliminary versions of our sampling exhaustiveness protocol have been already used in several integrative modeling applications (9)(10)(11)(61)(62)(63). Earlier, sampling exhaustiveness for integrative modeling was estimated, at best, by manual visual inspection of localization densities of clusters (7,8,44).…”

Hydrogen exchange protection factors can be extracted from sparse HDX-MS data

“…Results are most often reported as deuterium uptake curves (measured as centroid mass) or "butterfly charts" and thus provide only qualitative information at the resolution level of several amino acids. Limited attempts to extract rates as single residue level have been previously presented (29,30).…”

“…In contrast to previous methods (29,30), we chose to generate a large number of solutions and perform a thorough statistical analysis of them. The median and interquartile range provide information on residues for which a safe prediction of the protection factors can be made, and those for which it cannot.…”

Structural Interpretation of Hydrogen-Deuterium Exchange with Maximum-Entropy Simulation Reweighting

“…In practice, HDX-MS experiments measure deuteration averaged over lengthy peptide fragments rather than at the single-residue level. Even with robust methods to derive highresolution protection factors directly from experimental data (30,31), interpretation of the observed data in structural terms is not straightforward. Oftentimes HDX levels are colorcoded and mapped on known protein structures, which allows an intuitive visualization of the results and highlights dynamic or solvent-exposed protein regions.…”