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

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 Å 2 and 808 ± 2 Å 2 . [M – 4H] 4− ions were comprised of more compact (Ω = 676 ± 3 Å 2 ) and diffuse (i.e., more elongated, Ω = 779 ± 3 Å 2 ) ion conformer types. Ions were subjected to HDX in the drift tube using D 2 O 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 subchains. 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. Graphical Abstract ᅟ