Past experimental results and molecular dynamics simulations provide evidence that, under some conditions, electrospray ionization (ESI) of biomolecules produces ions that retain elements of solution phase structures. However, there is a dearth of information regarding the question raised by Breuker and McLafferty, "for how long, under what conditions, and to what extent, can solution structure be retained without solvent?" (Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18145). Here, we use cryogenic ion mobility-mass spectrometry to experimentally probe the structural evolution of the undecapeptide substance P (SP) during the final stages of ESI. The results reveal that anhydrous SP conformers originate from evaporation of cluster ions, specifically, [SP + 2H](2+) (H2O)n (n = 0 to ∼50) and [SP + 3H](3+) (H2O)n (n = 0 to ∼30), and that major structural changes do not occur during the evaporative process. In the case of [SP + 3H](3+), the results demonstrate that a compact dehydrated conformer population can be kinetically trapped on the time scale of several milliseconds, even when an extended gas phase conformation is energetically favorable.

Molecular dynamics (MD) simulations are used to model changes in the conformational preferences of a model peptide during the transition from a hydrated environment (charged nanodroplet generated by electrospray ionization) to the solvent-free peptide ion. The charged droplet consists of ∼2400 water molecules, 22 hydronium ions, and 10 chloride and contains a single Substance P (SP) [SP + 3H] ion (SP; amino acid sequence RPKPQQFFGLM-NH). Initially, droplet shrinkage involves a combination of solvent evaporation and ejection of excess charge, primarily hydronium ions. Further droplet shrinkage leads to a series of fission events, which includes the loss of some Cl ions. SP ions adapt to the smaller size droplet through small conformational changes that result in coiling of the hydrophobic C-terminus of the peptide on or near the droplet surface, intramolecular interactions involving the hydrophilic N-terminus of the peptide, and water-mediated interactions between the SP ion and HO and Cl ions. Calculated collision cross sections (CCS) for SP ions at various stages of desolvation are consistent with the results obtained from cryogenic ion mobility-mass spectrometry (cryo-IM-MS) measurements. Specifically, early in the decay of the charged droplet SP ions favor an extended conformation, whereas a compact conformer is favored during the final stages of dehydration.

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.

Biomolecular folding often occurs through a cooperative two-state reactant ↔ product transition; the term cooperative does not convey that intermediate structures are nonexistent but rather that these states are not observable by existing experimental techniques. Because of this, few intermediates have been studied and characterized. Recently, ion mobility spectrometry (IMS) measurements revealed that the oligomer polyproline-13 (Pro13, which in propanol (PrOH) favors the right-handed helical PPI structure having adjacent pyrrolidine rings in a cis configuration) folds through six sequential long-lived intermediates as it converts to the all-trans-configured PPII structure that is favored in aqueous solutions. Here, we examine the PPI → PPII folding transition for a HisPro13 sequence, i.e., Pro13 having a single histidine residue added to the N-terminus. Remarkably, the IMS measurements show that, upon addition of histidine, all of the IMS peaks associated with intermediate structures disappear. Instead, HisPro13 folds via a cooperative two-state transition, delayed by a significant induction period. The induction period is temperature dependent-shifting the transition to longer times at lower temperatures. Equilibrium studies show that the HisPro13 PPI → PPII transition is endothermic but favored entropically. From these clues, we propose a sequential folding mechanism and develop a model that suggests that ∼13-17 long-lived intermediates are likely responsible for the induction period. In this model, intermediates are separated by average individual activation barriers of ∼90 kJ·mol, and are entropically favorable.

Electron capture dissociation mass spectrometry offers several advantages for the analysis of peptides, most notably that backbone c and z fragments typically retain labile modifications such as phosphorylation. We have shown previously that, in some cases, the presence of phosphorylation has a deleterious effect on peptide sequence coverage, and hypothesized that intramolecular interactions involving the phosphate group were preventing separation of backbone fragments. In the present work, we seek to rationalize the observed ECD behavior through a combination of ECD of model peptides, traveling wave ion mobility mass spectrometry and molecular dynamics simulations. The results suggest that for doubly protonated ions of phosphopeptide APLpSFRGSLPKSYVK a salt-bridge structure is favored, whereas for the doubly-protonated ions of APLSFRGSLPKpSYVK ionic hydrogen bonds predominate.Graphical AbstractᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-015-1094-1) contains supplementary material, which is available to authorized users.