Gas-phase ion/ion chemistry for structurally sensitive probes of gaseous protein ion structure: Electrostatic and electrostatic to covalent cross-linking

Abstract

Intramolecular interactions within a protein are key in maintaining protein tertiary structure and understanding how proteins function. Ion mobility-mass spectrometry (IM-MS) has become a widely used approach in structural biology since it provides rapid measurements of collision cross sections (CCS), which inform on the gas-phase conformation of the biomolecule under study. Gas-phase ion/ion reactions target amino acid residues with specific chemical properties and the modified sites can be identified by MS. In this study, electrostatically reactive, gas-phase ion/ion chemistry and IM-MS are combined to characterize the structural changes between ubiquitin electrosprayed from aqueous and denaturing conditions. The electrostatic attachment of sulfo-NHS acetate to ubiquitin via ion/ion reactions and fragmentation by electron-capture dissociation (ECD) provide the identification of the most accessible protonated sites within ubiquitin as the sulfonate group forms an electrostatic complex with accessible protonated side chains. The protonated sites identified by ECD from the different solution conditions are distinct and, in some cases, reflect the disruption of interactions such as salt bridges that maintain the native protein structure. This agrees with previously published literature demonstrating that a high methanol concentration at low pH causes the structure of ubiquitin to change from a native (N) state to a more elongated A state. Results using gas-phase, electrostatic cross-linking reagents also point to similar structural changes and further confirm the role of methanol and acid in favoring a more unfolded conformation. Since cross-linking reagents have a distance constraint for the two reactive sites, the data is valuable in guiding computational structures generated by molecular dynamics. The research presented here describes a promising strategy that can detect subtle changes in the local environment of targeted amino acid residues to inform on changes in the overall protein structure. File list (2) download file view on ChemRxiv webb_electrostatic_x-link_manuscript.pdf (1.05 MiB) download file view on ChemRxiv Supplemental information.pdf (0.93 MiB)

Experimental Determination of Activation Energies for Covalent Bond Formation via Ion/Ion Reactions and Competing Processes

“…Covalent reactions and proton transfer gas-phase ion/ion chemistries between multiply charged cations and “sticky” anionic reagents are known to proceed through the formation of long-lived intermediates stabilized by strong electrostatic interactions. ,,,, Therefore, reaction rates of the covalent modification reactions, observed via neutral loss of the leaving group and proton transfer from the polypeptide to the reagent, can be readily probed by slow-heating methods. , The reagent used for this study was sulfo-benzoyl-HOAt, shown to be favored on kinetic grounds to proton transfer with only a small van der Waals barrier between the nascent covalent product complex and loss of the HOAt leaving group . We have recently used this reagent to probe gas-phase structures of ubiquitin cations formed from aqueous and low-pH methanolic solutions. , Since it is of interest to differentiate between gas-phase structures resulting directly from dehydration of the ions from droplets in the electrospray process and structures produced by activation and refolding of proteins into additional structures (i.e., CIU), the activation enthalpies, entropies, and free energies for the covalent and proton transfer reactions are valuable for characterizing the kinetics and mechanisms of these reactions versus CIU processes.…”

“…10 We have recently used this reagent to probe gas-phase structures of ubiquitin cations formed from aqueous and low-pH methanolic solutions. 12,38 Since it is of interest to differentiate between gas-phase structures resulting directly from dehydration of the ions from droplets in the electrospray process and structures produced by activation and refolding of proteins into additional structures (i.e., CIU), the activation enthalpies, entropies, and free energies for the covalent and proton transfer reactions are valuable for characterizing the kinetics and mechanisms of these reactions versus CIU processes. The ion/ion reaction products of ubiquitin 6 + ions and sulfo-benzoyl-HOAt 1 − ions formed in the trap cell with the application of various collision energies in the transfer cell are shown in Figure 2.…”

Multiplexed Conformationally-Selective, Localized Gas-Phase Hydrogen Deuterium Exchange of Protein Ions Enabled by Transmission-Mode Electron Capture Dissociation

“…Instrumental details have been described previously. [29][30][31][32]42 The NanoLockspray source was used with proteins introduced by nanoelectrospray ionization (nESI) from a pulled borosilicate glass capillary (P97 Flaming/Brown Micropipette puller, Sutter Instrument Company, Novato, CA) with a platinum wire inserted through the distal end of the capillary held at a positive potential of ~1 kV. Various charge states of the proteins produced by nESI were mass selected in the quadrupole for reaction with ND3 in the trap cell (Figure 1).…”

“…The lack of a solvent system serves to strengthen salt bridges in the gas phase and salt bridges are expected to be the final structural motifs from solution that are lost. 60 ECD 32 and ultraviolet photodissociation (UVPD) 61 have been previously used to assign protonation sites of native-like ubiquitin structures. Notably, both ECD and UVPD showed that lysine 33 is protonated for the 6 + ion, while UVPD and examination of ECD of the undeuterated 5 + ions showed that lysine 33 was not protonated for the 5 + ions, since the charge state of the fragments does not increase between z42 2+ and z45 2+ (i.e., between residues glycine 35 and lysine 29, respectively).…”

“…The exquisite selectivity of IM/MS/MS (or MS/IM/MS) measurements allows them to be uniquely capable of providing conformationally-selective structural measurements of heterogeneous and dynamic protein systems. 28 To gain localized three-dimensional structural information, it is desirable to couple a labeling technique with structurally selective IM/tandem MS. To this end, approaches for covalent [29][30][31] and noncovalent 32 irreversible labeling in the gas-phase have been coupled with MS/IM/MS workflows. In addition, gas-phase HDX has been used in various mass analyzers [33][34][35][36] and ion optics [37][38] towards threedimensional structural determination.…”