Investigation of Charge-State-Dependent Compaction of Protein Ions with Native Ion Mobility–Mass Spectrometry and Theory

Abstract

The precise relationship between native gas-phase protein ion structure, charge, desolvation, and activation remains elusive. Much evidence supports the Charge Residue Model for native protein ions formed by electrospray ionization, but scaling laws derived from it relate only to overall ion size. Closer examination of drift tube CCSs across individual native protein ion charge state distributions (CSDs) reveals deviations from global trends. To investigate whether this is due to structure variation across CSDs or contributions of long-range charge–dipole interactions, we performed in vacuo force field molecular dynamics (MD) simulations of multiple charge conformers of three proteins representing a variety of physical and structural features: β-lactoglobulin, concanavalin A, and glutamate dehydrogenase. Results from these simulated ions indicate subtle structure variation across their native CSDs, although effects of these structural differences and long-range charge-dependent interactions on CCS are small. The structure and CCS of smaller proteins may be more sensitive to charge due to their low surface-to-volume ratios and reduced capacity to compact. Secondary and higher order structure from condensed-phase structures is largely retained in these simulations, supporting the use of the term “native-like” to describe results from native ion mobility–mass spectrometry experiments, although, notably, the most compact structure can be the most different from the condensed-phase structure. Collapse of surface side chains to self-solvate through formation of new hydrogen bonds is a major feature of gas-phase compaction and likely occurs during the desolvation process. Results from these MD simulations provide new insight into the relationship of gas-phase protein ion structure, charge, and CCS.

Application of Multiple Length Cross-linkers to the Characterization of Gaseous Protein Structure

“…Indeed, an increase in the temperature (i.e., through collisional activation) of compact cytochrome c structures in the gas phase with cyclic tandem mobility experiments and trapped ion mobility experiments does produce extended states that do not compact down to the original CCS values, with the compact structures displaying a “permanent” increase in CCS. , With these considerations, the most accurate modeling of gaseous structures of well-folded proteins would need to keep temperatures low enough that the gas-phase rearrangement barriers are not reached. Additionally, using the crystal structure of folded proteins, followed by relaxation in the gas phase, accurately reproduces experimental observables. , …”

“…Additionally, using the crystal structure of folded proteins, followed by relaxation in the gas phase, accurately reproduces experimental observables. 15,40 ■…”

“…The processed data was exported as .mgf (Mascot Generic Format) files, and c and z fragments were annotated using LCMS spectator 33 (https://omics.pnl.gov/software/lcmsspectator), using a mass error tolerance of 20 ppm 34 were simulated using an existing protocol for gas-phase protein molecular dynamics (MD) that has been validated against experimental ion mobility measurements. 15,40 Briefly, five replicates of MD run for 100 ns in GROMACS 2019.4 41 using the GROMOS 43a2 force field 42 were simulated, incorporating distance restraints with a force constant of 100 kJ/mol Å 2 . 27 Charged sites were determined from ECD of cytochrome c 7 + (vide infra) and acidic residues were protonated (neutral) for simulations.…”

“…Thus, for the N ζ –N ζ distance restraint, 6.4 Å (two Lys “salt bridge” distances) was added to the cross-linker length while the N ζ –C ζ restraint was calculated by adding the cross-linker length and 7.4 Å (sum of Arg and Lys “salt bridge” distances, Figures and ). Gas-phase structures of cytochrome c were simulated using an existing protocol for gas-phase protein molecular dynamics (MD) that has been validated against experimental ion mobility measurements. , Briefly, five replicates of the MD run for 100 ns in GROMACS 2019.4 using the GROMOS 43a2 force field were simulated, incorporating distance restraints with a force constant of 100 kJ/mol Å 2 . Since 1,3-BDSA and 1,5-NDSA are structurally rigid, a restraint tolerance of ±0.1 Å (i.e., the distance where the potential in the restraint is minimized) was used during the MD simulations.…”

Ion Mobility–Mass Spectrometry Reveals the Structures and Stabilities of Biotherapeutic Antibody Aggregates

“…We note that while our models indicate that a significant amount of gas-phase compaction has occurred in our mAb ions when compared to other native globular proteins, the extent of compaction computed remains consistent with recent MD simulations aimed at tracking the details of this phenomenon. 61,62 In Figure 5C, we project the smallest (blue), median (green), and largest (orange) IgG models from the solution phase (top row) and gas phase (bottom row). It can be clearly seen in all three structures that there is considerable compaction of the hinge region.…”