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

Abstract

Hydrogen exchange technology provides a uniquely powerful instrument for measuring protein structural and biophysical properties, quantitatively and in a nonperturbing way, and determining how these properties are implemented to produce protein function. A developing hydrogen exchange-mass spectrometry method (HX MS) is able to analyze large biologically important protein systems while requiring only minuscule amounts of experimental material. The major remaining deficiency of the HX MS method is the inability to deconvolve HX results to individual amino acid residue resolution. To pursue this goal we used an iterative optimization program (HDsite) that integrates recent progress in multiple peptide acquisition together with previously unexamined isotopic envelope-shape information and a site-resolved back-exchange correction. To test this approach, residue-resolved HX rates computed from HX MS data were compared with extensive HX NMR measurements, and analogous comparisons were made in simulation trials. These tests found excellent agreement and revealed the important computational determinants.

Keywords: HDX-MS; isotope pattern; protein biophysics.

Fig. 1.

Overlapping peptides and site resolution. Peptides are indicated as horizontal bars. Inset represents an idealized ECD/ETD peptide dataset. The main diagram is a real peptide distribution for SNase obtained by proteolytic fragmentation. The naturally grouped subregions (within vertical dotted lines) are separately globally fit in the HDsite calculation. Site resolution implicit in the SNase peptide set is indicated by red dots for resolvable sites and blue rectangles for neighboring unresolved “switchable” sites, which could be resolved by obtaining more peptides, for example by using additional proteases.

Fig. 2.

Peptide mass spectra. Mass spectra are simulated for a 10-residue peptide with an average total of four bound deuterons, therefore the same centroid mass (Δm = 4) but with different unresolved D-occupancy distributions. Insets specify the averaged population D occupancies. (A) All residues are 50% D population average. (B) Four residues are H, four residues are D. (C) Four residues are 10% D, four residues are 90% D.

Fig. 3.

Toward amino acid resolution. (A) Some residue-resolved HX MS results for SNase compared with HX NMR results (dashed curves) or with the calculated rate for an unprotected amide not measured by NMR (dotted curve). (B) Comparison of HX NMR and HX MS data for SNase plotted in terms of HX protection factor (Pf = measured HX rate/expected unprotected rate). Filled symbols indicate directly determined HX MS D occupancy. Open symbols, switchable sites due to incomplete MS peptide overlap, are paired with their apparent NMR identities (this does not alter the fit quality in B and C). Dotted lines show deviations of threefold and 10-fold from the identity line. (C) Population distribution of site-resolved protection factors computed from HX MS data versus measured by NMR.

Fig. 4.

HDsite D-occupancy fitting results. (A–D) (Upper) The simulated peptide fragment datasets used. (A–D) (Lower) Results from five independent fitting runs for each peptide set with D occupancies and noise randomly assigned. The HDsite calculation was based on either centroid data (blue) or envelope shape data (red).

Fig. 5.

The course of iterative trial D-occupancy calculations. Twenty independent optimizations with random start points were run for the simulated peptide set in A. The peptide set is complete, implicitly discriminates every residue, and the number of peptides is not limiting relative to the number of sites to be fit, unlike the case in Fig. 4D. The RMSD for computed D occupancies (trial – true; y axis) is plotted against the RMSD for fitting to the input data (trial – experimental; x axis), where the experimental input is either the peptide centroids (B) or the isotopic peak amplitudes (C), as suggested in the Insets. The exploded view (D) more clearly illustrates the convergence of the envelope method results to the same D occupancies. Colors are used to distinguish the different runs.

Fig. 6.

Flow of the HDsite method. (Upper) Overall flow; (Lower) back-exchange treatment.