Automated data reduction for hydrogen/deuterium exchange experiments, enabled by high-resolution Fourier transform ion cyclotron resonance mass spectrometry

Abstract

Mass analysis of proteolytic fragment peptides following hydrogen/deuterium exchange offers a general measure of solvent accessibility/hydrogen bonding (and thus conformation) of solution-phase proteins and their complexes. The primary problem in such mass analyses is reliable and rapid assignment of mass spectral peaks to the correct charge state and degree of deuteration of each fragment peptide, in the presence of substantial overlap between isotopic distributions of target peptides, autolysis products, and other interferant species. Here, we show that at sufficiently high mass resolving power (m/Delta m(50%) > or = 100,000), it becomes possible to resolve enough of those overlaps so that automated data reduction becomes possible, based on the actual elemental composition of each peptide without the need to deconvolve isotopic distributions. We demonstrate automated, rapid, reliable assignment of peptide masses from H/D exchange experiments, based on electrospray ionization FT-ICR mass spectra from H/D exchange of solution-phase myoglobin. Combined with previously demonstrated automated data acquisition for such experiments, the present data reduction algorithm enhances automation (and thus expands generality and applicability) for high-resolution mass spectrometry-based analysis of H/D exchange of solution-phase proteins.

Figure 1

ESI FT-ICR mass spectra, showing five myoglobin peptide ions 910 < m/z < 950. Top: blank control experiment (no exposure to D₂O). Peaks a–d for F₁₃₈RNDIAAKYKELGFQG₁₅₃ (2+ charge state) represent the natural abundance isotopic distribution (a = monoisotopic, and b–d contain 1–3 ¹³C plus ¹⁵N atoms. Bottom: same display, but for peptides isolated after 2 min of H/D exchange.

Figure 2

Resolution and identification of pepsin-digested myoglobin peptides in the absence of H/D exchange. Top: mass spectrum showing resolution of the isotopic distributions of a doubly charged target peptide (a, b, c, d) from another quadruply-charged peptide (filled circle). Bottom: resolution of the overlapped isotopic distributions of the same doubly charged target peptide (a, b, c, d) and an interferant singly-charged molecule (asterisk).

Figure 3

Bottom: ESI FT-ICR mass spectral segment showing the isotopic distribution for a doublycharged pepsin-digested myoglobin fragment peptide. Top: simulated (heavy lines) and experimental (thin line) spectra for the mass sub-window spanning unit m/z range. The outer pair of dotted vertical lines in the top diagram define the width of the m/z “sub-window” for peak searching (see text).

Figure 4

ESI FT-ICR mass spectra of a 5+ (top) and 6+ (bottom) pepsin-digested myoglobin peptide fragments. In each case, the target peptide (top inset, blue; bottom inset, red) is selected over an interferant peptide (black in each inset) based on closer match to the sub-window midpoint for that nominal mass (see text).

Figure 5

Deuterium uptake profiles for each of two triplycharged similar-length peptides produced by pepsin digestion of myoglobin. The HDX analysis is consistent with the surface accessibility of each peptide segment (see text).