Computing H/D-Exchange rates of single residues from data of proteolytic fragments

Abstract

Background: Protein conformation and protein/protein interaction can be elucidated by solution-phase Hydrogen/Deuterium exchange (sHDX) coupled to high-resolution mass analysis of the digested protein or protein complex.In sHDX experiments mutant proteins are compared to wild-type proteins or a ligand is added to the protein andcompared to the wild-type protein (or mutant). The number of deuteriums incorporated into the polypeptidesgenerated from the protease digest of the protein is related to the solvent accessibility of amide protons withinthe original protein construct.Results: In this work, sHDX data was collected on a 14.5 T FT-ICR MS. An algorithm was developed based oncombinatorial optimization that predicts deuterium exchange with high spatial resolution based on the sHDX dataof overlapping proteolytic fragments. Often the algorithm assigns deuterium exchange with single residueresolution.Conclusions: With our new method it is possible to automatically determine deuterium exchange with higherspatial resolution than the level of digested fragments