Ion Mobility Spectrometry-Hydrogen Deuterium Exchange Mass Spectrometry of Anions: Part 3. Estimating Surface Area Exposure by Deuterium Uptake

Abstract

Gas-phase Hydrogen deuterium exchange (HDX), collision cross section (CCS) measurement, and molecular dynamics simulation (MDS) techniques were utilized to develop and compare three methods for estimating the relative surface area exposure of separate peptide chains within bovine insulin ions. Electrosprayed [M−3H]3− and [M−5H]5− insulin ions produced a single conformer type with respective collision cross sections of 528 ± 5 Å2 and 808 ± 2 Å2. [M−4H]4− ions were comprised of more compact (Ω = 676 ± 3 Å2) and diffuse (i.e., more elongated, Ω = 779 ± 3 Å2) ion conformer types. Ions were subjected to HDX in the drift tube using D2O as the reagent gas. Collision-induced dissociation was used to fragment mobility-selected, isotopically labeled [M−4H]4− and [M−5H]5− ions into the protein sub-chains. Deuterium uptake levels of each chain can be explained by limited inter-chain isotopic scrambling upon collisional activation. Using nominal ion structures from MDS and a hydrogen accessibility model, the deuterium uptake for each chain was correlated to its exposed surface area. In separate experiments the per-residue deuterium content for the protonated and deprotonated ions of the synthetic peptide KKDDDDDIIKIIK were compared. The differences in deuterium content indicated the regional HDX accessibility for cations versus anions. Using ions of similar conformational type, this comparison highlights the complementary nature of HDX data obtained from positive- and negative-ion analysis.

Comprehensive Gas-Phase Peptide Ion Structure Studies Using Ion Mobility Techniques: Part 2. Gas-Phase Hydrogen/Deuterium Exchange for Ion Population Estimation

“…Previous experiments have shown that per-residue HDX values can provide information about the relative distances of heteroatom sites to charge sites and deuterium incorporation sites, the surface accessibility of residues, and the charge site configuration of the peptide ions [8, 20, 25, 26]. In this manuscript, HAS scoring coupled with a NEC model is implemented for each candidate structure to produce its theoretical per-residue uptake pattern.…”

“…To address this problem, in separate studies, the contribution of each residue to the HDX rate for select conformer types was measured; using the same distance model, theoretical contributions to rate better approximated measured contributions for in - silico structures intended to represent conformer type [20]. Because measuring the contributions to rate constants at the individual amino acid residue level is an extremely time-intensive process and difficult to obtain for larger ions, an effective collision model was proposed to describe deuterium uptake levels at different partial pressures of D 2 O [25, 26] for select ion conformers.…”

Comprehensive Peptide Ion Structure Studies Using Ion Mobility Techniques: Part 1. An Advanced Protocol for Molecular Dynamics Simulations and Collision Cross-Section Calculation

“…To begin addressing these issues, gas-phase HDX has been introduced as a second criterion for structural characterization [27-29, 61, 62]. In the second installment of this work, comparisons of the experimental deuterium uptake values and the hypothetical values (obtained from a modified algorithm assessing the accessibility of hydrogens on candidate structures) are used to improve conformer selection and to assign relative population values along a MD trajectory.…”

Interpreting Hydrogen–Deuterium Exchange Events in Proteins Using Atomistic Simulations: Case Studies on Regulators of G-Protein Signaling Proteins

“…35,37,43,44 Therefore, HDX rates implicitly involve structural changes and dynamics in proteins. 29 A variety of models have been used to determine protein conformational states using Monte Carlo (MC) 38,45 or molecular dynamics (MD) 29–31,39,40,42,46–54 approaches.…”