Increasing Ubiquitin Ion Resistance to Unfolding in the Gas Phase Using Chloride Adduction: Preserving More “Native-Like” Conformations Despite Collisional Activation

Abstract

Electrospray ionization (ESI) of ubiquitin from acidified (0.1%) aqueous solution produces abundant ubiquitin-chloride adduct ions, [M + nH + xCl]((n - x)+), that upon mild heating react via elimination of neutral HCl. Ion mobility collision cross section (CCS) measurements show that ubiquitin ions retaining chloride adducts exhibit CCS values similar to those of the "native-state" of the protein. Coupled with results from recent molecular dynamics (MD) simulations for the evolution of a salt-containing electrospray droplet, this study provides a more complete picture for how the presence of salts affects the evolution of protein conformers in the final stages of dehydration of the ESI process and within the instrument.

Experimental and theoretical investigation of overall energy deposition in surface-induced unfolding of protein ions

“…These data indicate that in CIU ions retain a high internal temperature or already have many of their native non-covalent interactions disrupted as they enter the Trap. Decreased adduction following in-source activation may also contribute to the observed destabilization, as adducts have been shown to have a slight stabilizing effect in CIU,58–61 and in-source activation in these experiments dissociates approximately 225 Da of adducts. However, for SIU, in-source activation has no significant effect on unfolding appearance energies, suggesting that these ions cool prior to SIU.…”

Collision-Induced Unfolding of Partially Metalated Metallothionein-2A: Tracking Unfolding Reactions of Gas-Phase Ions

“…Combining ion mobility with nESI-MS, e.g., nESI-IM-MS, affords direct measurements of the conformational diversity of the ion population as well as ion size (shape), information that can be related to structure and conformation. 21,28,29,46 Apo-MT has been described as an intrinsically disordered protein, and indeed, the CCS profiles for 6+ and 5+ ions are composed of multiple peaks (Figure S1), consistent with a heterogeneous population of states, as opposed to a population of interconverting states. Note also that CCS profiles obtained from methanolic solutions, generally thought to be denaturing conditions, are very similar, suggesting that the addition of methanol does not significantly alter the structural or conformational diversity of MT.…”

“…16 Nano ESI-CIU-MS experiments were performed as described previously. 29 Identical collisional heat minimizing conditions were used prior to collisional activation. The ion of interest was mass-selected by the quadrupole mass analyzer and then collisionally activated (CA) prior to IM analysis.…”

Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters

“…Charge interactions with lipid head groups are maintained during collisional activation of the protein and provide local stabilization via multiple H-bonds. This is analogous to the protective effects of metal ions on proteins in the gas phase, where multiply coordinated ions reduce the build-up of destabilizing structural rearrangements that occur during collisional activation2627. Unlike metal ions, however, which have a high propensity to form non-specific adducts during desolvation, the complexes between NapA and phospholipids are formed inside detergent micelles in solution, exhibiting an intermediate degree of selectivity for charged lipids bound to a subset of annular positions28.…”