High-Resolution Hydrogen-Deuterium Protection Factors from Sparse Mass Spectrometry Data Validated by Nuclear Magnetic Resonance Measurements

Experimental measurement of time-dependent sponta-neous exchange of amide protons with deuterium of the solventprovides information on the structure and dynamical structuralvariation in proteins. Two experimental techniques are used to probethe exchange: NMR, which relies on different magnetic properties ofhydrogen and deuterium, and MS, which exploits the change in massdue to deuteration. NMR provides residue-specific information, thatis, the rate of exchange or, analogously, the protection factor (i.e., theunitless ratio between the rate of exchange for a completelyunstructured state and the observed rate). MS provides informationthat is specific to peptides obtained by proteolytic digestion. Thespatial resolution of HDX-MS measurements depends on theproteolytic pattern of the protein, the fragmentation method used,and the overlap between peptides. Different computational approacheshave been proposed to extract residue-specific information from peptide-level HDX-MS measurements. Here, we demonstrate theadvantages of a method recently proposed that exploits self-consistency and classifies the possible sets of protection factors into afinite number of alternative solutions compatible with experimental data. The degeneracy of the solutions can be reduced (orcompletely removed) by exploiting the additional information encoded in the shape of the isotopic envelopes. We show how sparseand noisy MS data can provide high-resolution protection factors that correlate with NMR measurements probing the same proteinunder the same conditions.

Automated summary

This study investigates the structure and dynamic structural variation in proteins through the experimental measurement of the spontaneous exchange of amide protons with deuterium in the solvent. Two experimental techniques, NMR and MS, are used to probe the exchange. NMR provides residue-specific information, while MS provides information specific to proteolytic digestion products. Different computational approaches have been proposed to extract residue-specific information from peptide-level MS data.