Dueling electrospray implemented on a traveling-wave ion mobility/time-of-flight mass spectrometer: Towards a gas-phase workbench for structural biology

Webb, Ian; Morrison, Lindsay; Brown, Jeffery M.

doi:10.1016/j.ijms.2019.116177

“…1 C), proton transfer becomes favorable as the transition state is more disordered than the transition state for covalent bond formation, evidenced by a positive activation entropy for proton transfer versus a negative activation entropy for covalent bond formation. This reflects the ability in previous experiments to tune the reaction conditions in the trap traveling wave cell appropriately 12,32 such that covalent bond formation was the only observed ion/ion product, ensuring that intramolecular proton transfer to sulfo-benzoyl HOAt does not measurably occur, in agreement with the measurements here that covalent bond formation is the kinetically favored product.…”

Section: Covalent and Proton Transfer Reactionssupporting

confidence: 86%

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

Kit

¹

,

Shepherd

²

,

Prell

³

et al. 2021

J. Am. Soc. Mass Spectrom.

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The combination of ion/ion chemistry with commercially available ion mobility/mass spectrometry systems has allowed rich structural information to be obtained for gaseous protein ions. Recently, the simple modification of such an instrument with an electrospray reagent source has allowed three-dimensional gas-phase interrogation of protein structures through covalent and non-covalent interactions coupled with collision cross section measurements. However, the energetics of these processes have not yet been studied quantitatively. In this work, previously developed Monte Carlo simulations of ion temperatures inside traveling wave ion guides are used to characterize the energetics of the transition state of activated ubiquitin cation/reagent anion long-lived complexes formed via ion/ion reactions. The ΔH ‡ and ΔS ‡ of major processes observed from collisional activation of long-lived gas phase ion/ion complexes, namely collision induced unfolding (CIU), covalent bond formation, or neutral loss of the anionic reagent via intramolecular proton transfer, were determined. Covalent bond formation via ion/ion complexes was found to be significantly lower energy compared to unfolding and bond cleavage. ΔG ‡ of activation of all three processes lie between 55 and 75 kJ/mol, easily accessible with moderate collisional activation. Bond formation is favored over reagent loss at lower activation energies, whereas reagent loss becomes competitive at higher collision energies. Though ΔG ‡ are between CIU of a precursor ion and covalent bond formation of its ion/ion product complex are comparable, our data suggest covalent bond formation does not require extensive isomerization, supporting evidence from previous structural studies that these ion/ion reactions measure compact gas phase structures. File list (2) download file view on ChemRxiv manuscript_final.pdf (547.56 KiB) download file view on ChemRxiv Supporting Information_final.pdf (177.83 KiB)

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“…cell (e-MSion, Corvallis, OR). Instrumental details for the ion/ion reactions 24,37 and ExD cell 33 have been described previously. The NanoLockspray source was used with proteins infused through the sample sprayer and anionic reagents through the reference sprayer..…”

Section: Methodsmentioning

confidence: 99%

“…22 Gas-phase ion/ion reactions have also been used to report on threedimensional structure of protein cations formed from native-like or denaturing solutions. [23][24][25] A combination of experimental 4,7,[26][27][28][29] and computational studies [30][31] have shown that under carefully-controlled experimental conditions, the solution structures of electrosprayed peptides and proteins is conserved upon electrospray ionization, making methods for probing gas-phase three-dimensional structures of proteins an extremely valuable addition to structural biology techniques.…”

Section: Introductionmentioning

confidence: 99%

“…However, the sites where the sulfo groups are attached electrostatically could not be determined since CID can lead to possible scrambling of the electrostatically-bound linker during backbone fragmentation. 25 In this study, we follow up on the previous results and implement an ion/ion, ion mobility/mass spectrometry (I 3 M 2 S) platform 24 to characterize the products of the gas-phase electrostatic ion/ion reaction between intact ubiquitin cation and the negatively charged sulfo-NHS based reagents (sulfo-NHS acetate and sulfo-EGS) with electron capture dissociation (ECD). 33 We observed the non-covalent complex, formed from electrostatic binding of the sulfonate group(s) of the anion to protonated residues from ubiquitin, and separate the reaction products by traveling wave ion mobility spectrometry (TWIMS) [34][35] .…”

Section: Introductionmentioning

confidence: 99%

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

Kit

¹

,

Carvalho

²

,

Vilseck

³

et al. 2021

International Journal of Mass Spectrometry

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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)

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“…All the experiments were performed on a Synapt G2-Si High Definition Mass Spectrometer (Waters Corporation, Wilmslow, U.K.), equipped with electron transfer dissociation (ETD), a NanoLockspray source, an external electrospray voltage control module (GAA Custom Electronics LLC, Kennewick, WA), and an ExD cell (e-MSion, Corvallis, OR). Instrumental details for the ion/ion reactions 24,37 and ExD cell 33 have been described previously. The NanoLockspray source was used with proteins infused through the sample sprayer and anionic reagents through the reference sprayer..…”

Section: Methodsmentioning

confidence: 99%

Gas-Phase Ion/Ion Chemistry for Structurally Sensitive Probes of Gaseous Protein Ion Structure: Electrostatic and Electrostatic to Covalent Cross-Linking

Kit¹,

Carvalho²,

Vilseck³

et al. 2021

Preprint

0

View full text Add to dashboard Cite

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.

show abstract

Dueling electrospray implemented on a traveling-wave ion mobility/time-of-flight mass spectrometer: Towards a gas-phase workbench for structural biology

Cited by 16 publications

References 54 publications

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

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

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

Gas-Phase Ion/Ion Chemistry for Structurally Sensitive Probes of Gaseous Protein Ion Structure: Electrostatic and Electrostatic to Covalent Cross-Linking

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