The Application of Native (Ion Mobility)-Mass Spectrometry to Probe the Structure of Proteins and Protein Complexes

  • Aidan France

Student thesis: Phd

Abstract

Native ion mobility-mass spectrometry (IM-MS) is a technique wherein proteins are introduced to mass spectrometers for analysis with as little perturbation to their solvated form as possible. Native IM-MS provides size and shape information for proteins from low volume (uL)/concentration samples (uM). IM-MS data can be used to describe conformational landscapes as CCS distributions and computational approaches permit comparisons between the structures sampled in vacuo and those present in solution. This thesis uses native IM-MS to examine protein structure. In Chapter 2, experiments on three IM-MS platforms with two drift gases (helium and nitrogen) evaluate the reproducibility of protein CCS measurements. Minimising ion activation is achieved with careful reference to the CCS distributions. CCS/CCS distributions are largely similar across instruments with nitrogen as a drift gas and vary more with helium, highlighting limitations of TWIMS CCS calibration as well as the inherent flexibility of proteins. In Chapters 3 and 4, native (IM)-MS is applied, for the first time, to the analysis of proteins directly from food extracts. The food industry is of great socioeconomic importance, therefore new tools which can quickly and unequivocally detect adulterants/allergens within foods are of great value. We have shown the utility of native (IM)-MS in the assignment of a significant proportion (up to ~90%) of the protein content of food substances of varying complexity. This analysis is fast (anywhere from 5 mins) and amenable to the detection of proteinaceous adulterants within beverages (as low as 1% v/v). The relative quantitation of proteoforms relating to natural abundances/processing effects is also demonstrated. In Chapter 5, an investigation is performed on the interaction of the neurotransmitter noradrenaline (NA) with alpha-synuclein (AS). Mass spectrometry is employed alongside other biophysical techniques to show that, upon incubation with NA, AS undergoes oxidation previously linked with oligomer formation. Our research permits a hypothesis that these higher-order species likely interact with NA oxidised derivatives (NAODs) to promote the formation of neurotoxic oligomers with notable anti-parallel beta-sheet structure.
Date of Award1 Aug 2020
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorRichard Unwin (Supervisor), Perdita Barran (Supervisor) & Garth Cooper (Supervisor)

Keywords

  • NA oxidised derivatives (NAODs)
  • Noradrenaline (NA)
  • alpha-synuclein (AS)
  • CCS distribution
  • Collision Cross Section (CCS)
  • Native ion mobility-mass spectrometry

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