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

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.

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

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.

Characterization of Protein Structure with Ion Mobility Mass Spectrometry, Multiplexed Fragmentation Strategies and Data Directed Analysis

<p>Activated ion mobility measurements provide Insights to the stability of tertiary and quaternary structures of proteins and pairing such approaches with fragmentation can delineate which part(s) of the primary sequence are disrupted from a folded structure. In this work we use 213 nm photodissociation coupled with ion mobility mass spectrometry and collisional activation to determine the conformational landscape of model proteins. UVPD experiments are performed on proteins following in source activation as well as on collisionally activated photoproducts post ion mobility separation. For cytochrome c, there is a significant increase in the fragmentation yield with collisional activation post mobility, for all conformational states. Similar strategies are deployed with the model multimeric proteins, concanavalin a, and haemoglobin. For these complexes’ CID leads to classic asymmetric charge distribution in subunit products, which when preceded by UV irradiation yields fragments from within the sub-unit that can be mapped to the quaternary fold. Data driven, multivariate analysis (MVA) was used to determine the significant differences in UVPD and CID fragmentation pattern following in source activation. This data driven approach reveals diagnostic fragments without <i>a priori</i> assignments limited to predicated backbone cleavage and provides a new approach to map conformation landscapes that may have wider utility.</p>

Characterization of Protein Structure with Ion Mobility Mass Spectrometry, Multiplexed Fragmentation Strategies and Data Directed Analysis

<p>Activated ion mobility measurements provide Insights to the stability of tertiary and quaternary structures of proteins and pairing such approaches with fragmentation can delineate which part(s) of the primary sequence are disrupted from a folded structure. In this work we use 213 nm photodissociation coupled with ion mobility mass spectrometry and collisional activation to determine the conformational landscape of model proteins. UVPD experiments are performed on proteins following in source activation as well as on collisionally activated photoproducts post ion mobility separation. For cytochrome c, there is a significant increase in the fragmentation yield with collisional activation post mobility, for all conformational states. Similar strategies are deployed with the model multimeric proteins, concanavalin a, and haemoglobin. For these complexes’ CID leads to classic asymmetric charge distribution in subunit products, which when preceded by UV irradiation yields fragments from within the sub-unit that can be mapped to the quaternary fold. Data driven, multivariate analysis (MVA) was used to determine the significant differences in UVPD and CID fragmentation pattern following in source activation. This data driven approach reveals diagnostic fragments without <i>a priori</i> assignments limited to predicated backbone cleavage and provides a new approach to map conformation landscapes that may have wider utility.</p>

Unimolecular Reactions

This chapter considers unimolecular reactions; photo-induced reactions, that is, true unimolecular reactions; and reactions initiated by collisional activation, that is, apparent unimolecular reactions where it is assumed that the time scales for activation and subsequent reaction are well separated. Elements of classical and quantum dynamical descriptions are discussed, including Slater theory and the quantum mechanical description of photo-induced reactions. Statistical theories aiming at the calculation of micro-canonical as well as canonical rate constants are discussed, including a detailed discussion of RRKM theory. It concludes with a discussion of femtochemistry, that is, the observation and control of chemical dynamics using femtosecond pulses of electromagnetic radiation, focusing on the control of unimolecular reactions via the interaction with coherent light; that is, laser control.