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

Abstract

BackgroundProtein 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 and compared to the wild-type protein (or mutant). The number of deuteriums incorporated into the polypeptides generated from the protease digest of the protein is related to the solvent accessibility of amide protons within the original protein construct.ResultsIn this work, sHDX data was collected on a 14.5 T FT-ICR MS. An algorithm was developed based on combinatorial optimization that predicts deuterium exchange with high spatial resolution based on the sHDX data of overlapping proteolytic fragments. Often the algorithm assigns deuterium exchange with single residue resolution.ConclusionsWith our new method it is possible to automatically determine deuterium exchange with higher spatial resolution than the level of digested fragments.

Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis

“…We find that the effort to achieve residue resolution is not limited by a difficult calculational barrier (17,18,20) but by experimental difficulties. To reach this goal, it is necessary to obtain many sequentially overlapping peptide fragments that cover the experimental protein several times over.…”

“…More penetrating conclusions could be drawn if it were possible to extend structural resolution to the individual amino acid level. Recently reported efforts have moved in this direction (15,(17)(18)(19)(20)(21)(22)(23)(24).…”

“…However, in the absence of a common terminus, the D occupancy of the overhanging residues cannot be obtained by simple subtraction of the D-dependent mass centroids of neighboring peptides, as for the ECD/ETD dataset. Site-resolution analysis must consider simultaneously the multiple distributed segments and overhangs in the entire peptide collection (15,(17)(18)(19)(20). We find that this kind of analysis can be greatly improved by the use of an aspect of the MS data that has been ignored before, namely the multiple peaks in the isotopic envelope of each peptide fragment rather than just the single parameter mass centroid.…”

Improved Sequence Resolution by Global Analysis of Overlapped Peptides in Hydrogen/Deuterium Exchange Mass Spectrometry

“…We have an option of estimating the rates for these "fast" and "slow" residues in the initial search of first and final incubation points, after which their rates are allowed to vary ± 25 %, but in practice the program is fast enough to float even those residues. The black bars on top of Figure 2b denote five groups of linked residues (3, 4), (6,7), (9)(10)(11)(12)(13)(14)(15), (16,17), and (27-29) for which we can determine the individual rate constants but cannot assign them to specific residues.…”

“…An elegant method to assign the rate constants to the overlapped peptide fragments was recently proposed by Althaus et al [16]. That routine begins from input rate constants determined by some other tool (e.g., maximum entropy method or integer linear programming) and assigns those to given residues by use of a combinatorial approach.…”