Category: Publications

Initial Steps of Amyloidogenic Peptide Assembly Revealed by Cold-Ion Spectroscopy

The early stages of fibril formation are difficult to capture in solution. We use cold‐ion spectroscopy to examine an 11‐residue peptide derived from the protein transthyretin and clusters of this fibre‐forming peptide containing up to five units in the gas phase. For each oligomer, the UV spectra exhibit distinct changes in the electronic environment of …

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Uncoupling conformational states from activity in an allosteric enzyme

ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)-L-alanine (TIH) as an allosteric activator …

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UV Photodissociation Mass Spectrometry Accurately Localize Sites of Backbone Deuteration in Peptides

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is now a routinely used technique to inform on protein structure, dynamics, and interactions. Localizing the incorporated deuterium content on a single residue basis increases the spatial resolution of this technique enabling detailed structural analysis. Here, we investigate the use of ultraviolet photodissociation (UVPD) at 213 nm to measure deuterium …

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Mobilising ion mobility mass spectrometry for metabolomics

Chromatography-based mass spectrometry approaches (xC-MS) are commonly used in untargeted metabolomics, providing retention time, m/z values and metabolite-specific fragments, all of which are used to identify and validate an unknown analyte. Ion mobility-mass spectrometry (IM-MS) is emerging as an enhancement to classic xC-MS strategies, by offering additional ion separation as well as collision cross section (CCS) determination. …

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Initial Protein Unfolding Events in Ubiquitin, Cytochrome c and Myoglobin Are Revealed with the Use of 213 nm UVPD Coupled to IM-MS

The initial stages of protein unfolding may reflect the stability of the entire fold and can also reveal which parts of a protein can be perturbed, without restructuring the rest. In this work, we couple UVPD with activated ion mobility mass spectrometry to measure how three model proteins start to unfold. Ubiquitin, cytochrome c and …

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ORIGAMI: A software suite for activated ion mobility mass spectrometry (aIM-MS) applied to multimeric protein assemblies

We present here a software suite (ORIGAMI) that facilitates the rapid acquisition and analysis of ion mobility data following collisional activation. ORIGAMI was developed for use on Waters Synapt instruments where data acquisition is achieved by interfacing WREnS (Waters Research Enabled Software) and MassLynx. Two components are presented, the first is ORIGAMIMS which enables the activation of ions via a …

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Molecular Insights into the Thermal Stability of mAbs with Variable-Temperature Ion-Mobility Mass Spectrometry

The aggregation of protein-based therapeutics such as monoclonal antibodies (mAbs) can affect the efficacy of the treatment and can even induce effects that are adverse to the patient. Protein engineering is used to shift the mAb away from an aggregation-prone state by increasing the thermodynamic stability of the native fold, which might in turn alter …

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Distinguishing Loss of Structure from Subunit Dissociation for Protein Complexes with Variable Temperature Ion Mobility Mass Spectrometry

Proteins and protein complexes undergo structural changes depending on the surrounding environment, in turn affecting their biological function.  Here, VT-MS is applied to study four multimeric protein complexes and allow for decoupling of their melting temperature (Tm) from the protein complex dissociation temperature (TGPD).   VT-IM-MS is used to investigate structural changes of these proteins at …

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Signature dynamics of intact immunoglobulin G revealed by drift-tube ion-mobility mass spectrometry and molecular modeling

Monoclonal antibodies (mAb) are a rapidly growing group of biopharmaceuticals.  Due to their intrinsic flexibility, intact mAbs provide a challenge with regards to higher order structure characterization.  Intact mAbs and their fragments are explored here using the DT-IM-MS and were found to be far more dynamic in the gas-phase than proteins of comparable size.  This …

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Insights into the Conformations of Three Structurally Diverse Proteins: Cytochrome c, p53, and MDM2, Provided by Variable-Temperature Ion Mobility Mass Spectrometry

Thermally induced conformational transitions of three proteins of increasing intrinsic disorder—cytochrome c, the tumor suppressor protein p53 DNA binding domain (p53 DBD), and the N-terminus of the oncoprotein murine double minute 2 (NT-MDM2)—have been studied by native mass spectrometry and variable-temperature drift time ion mobility mass spectrometry (VT-DT-IM-MS). Ion mobility measurements were carried out at temperatures …

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