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Browsing by Subject "Native mass spectrometry"
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Item Application of Multiple Length Crosslinkers to the Characterization of Gaseous Protein Structure(American Chemical Society, 2022-09-13) Kit, Melanie Cheung See; Webb, Ian K.; Chemistry and Chemical Biology, School of ScienceThe speed, sensitivity, and tolerance of heterogeneity of native mass spectrometry, as well as the kinetic trapping of solution-like states during electrospray, makes mass spectrometry an attractive method to study protein structure. Increasing resolution of ion mobility measurements and mass resolving power and range are leading to the increase of the information content of intact protein measurements, and an expanded role of mass spectrometry in structural biology. Herein, a suite of different length noncovalent (sulfonate to positively charged side chain) crosslinkers was introduced via gas-phase ion/ion chemistry and used to determine distance restraints of kinetically trapped gas-phase structures of native-like cytochrome c ions. Electron capture dissociation allowed for the identification of crosslinked sites. Different length linkers resulted in distinct pairs of side chains being linked, supporting the ability of gas-phase crosslinking to be structurally specific. The gas-phase lengths of the crosslinkers were determined by conformational searches and density functional theory, allowing for the interpretation of the crosslinks as distance restraints. These distance restraints were used to model gas-phase structures with molecular dynamics simulations, revealing a mixture of structures with similar overall shape/size but distinct features, thereby illustrating the kinetic trapping of multiple native-like solution structures in the gas phase.Item Dueling Electrospray Implemented on a Traveling-Wave Ion Mobility/Time-Of-Flight Mass Spectrometer: Towards a Gas-Phase Workbench for Structural Biology(Elsevier, 2019-07-05) Webb, Ian; Morrison, Lindsay; Brown, JeffThe traveling wave trap cell of a commercial ion mobility mass spectrometer (IM/MS) was used as a gas-phase reactor for covalent chemistry by making a simple modification to a standard nanoelectrospray source. Reagents and analytes were generated from pulsed opposite polarity nanoelectrospray sources and isolated by their m/z prior to reaction. Covalent bond formation was first observed with the model peptide angiotensin I. The modification site was identified as the N-terminus of the peptide by collision induced dissociation (CID). The IM cell separated the covalent reaction product from the proton transfer product by their respective ion mobilities. Next, the effects of several trapping parameters, including the trap traveling wave height, the trap RF voltage, and the trap pressure, were evaluated. Decreasing traveling wave height and increasing RF voltage and pressure increased the number of proton transfer events from apomyoglobin to reagent anions. The 6+ charge state of ubiquitin generated from nanospray under native-like conditions was covalently modified in the gas phase through ion/ion reactions. Probing the reacted protein with CID led to the assignment of lysine 29 and arginine 54 as reactive nucleophiles accessible to the reagent. IM analysis of the unmodified native-like 6+ charge state revealed that the gas-phase structure of the protein in the trap was in its compact form. Overall, we introduce a promising method for three-dimensional structural characterization of biomacromolecules.