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

Abstract

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)

Collision-Induced Unfolding of Native-like Protein Ions Within a Trapped Ion Mobility Spectrometry Device

“…While this charge state has been investigated several times in literature, none are capable of resolving the complexity acquired with this technique. [29][30][31] To find similar complexity in literature, previous data from the 7+ charge state must be examined. This charge state displays similar complexity but only three extended structures are annotated at the highest energies utilized, while six are easily annotated with the TIMS-based technique (Figure 1C).…”

Collision-Induced Unfolding of Native-like Protein Ions Within a Trapped Ion Mobility Spectrometry Device

“…While this charge state has been investigated several times in literature, none are capable of resolving the complexity acquired with this technique. [30][31][32] To find similar complexity in literature, previous data from the 7+ charge state must be examined. This charge state displays similar complexity but only three extended structures are annotated at the highest energies utilized, while six are easily annotated with the TIMS-based technique (Figure 1C).…”

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 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.…”