Probing Early Stage Aggregates of Amyloidogenic Proteins using Mass Spectrometry Based Methods

  • Ashley Phillips

Student thesis: Phd

Abstract

Mass Spectrometry (MS) and Ion Mobility - Mass Spectrometry (IM-MS) can be used to investigate protein structure and dynamics and are ideally positioned to study intrinsically disordered and amyloidogenic proteins, whose diverse conformational space and/or oligomeric state is hard to track accurately. This thesis uses hybrid MS approaches including IM-MS, Cross-linking IM-MS and ECD-FT-ICR MS to probe the structure of alpha-Synuclein and Amyloid-beta (Abeta). For alpha-Synuclein, the effect of solution pH and ionisation polarity on the species observed by MS and IM-MS is investigated. Conformational families observed by Cross-linking IM-MS provides a link between the solution and gas phase structures of alpha-Synuclein observed here and our data correlates with that reported by other groups. MS, IM-MS and HDX-MS are used to probe alpha-Synuclein during the early stages of aggregation. A specific aggregation competent conformer is not observed suggesting that the solution constituents remain conformationally dynamic. We observe shifts in the species observed by MS and IM-MS between samples and our data contributes to an array of conflicting structural studies indicating that alpha-Synuclein adopts a diverse range of species with significant variation.For Abeta(1-42) and Abeta(1-40) Collision Induced Unfolding and ETD/ETcaD demonstrate that Abeta(1-42) adopts a compact conformation bound by intramolecular interactions. Changes to the Abeta(1-42) and Abeta(1-40) ATDs following SID are correlated to known structure influencing intermolecular interactions and demonstrate the large structural difference between Abeta(1-42) and Abeta(1-40) despite differing by only two C-terminal amino acids. IM-MS is used to classify the mode of action of anti-aggregation drug candidates on Abeta(1-42). The anti-aggregation capacity of the retro-inverso peptide, RI-OR2 is shown to result from inducing the compaction or extension of Abeta(1-42), preventing the adoption of an aggregation competent structure. In contrast, the flavonoid Rutin is shown to act solely through inducing Abeta(1-42) compaction.This thesis demonstrates the power of MS based methods to investigate the diverse range of structures of intrinsically disordered aggregating proteins implicated in disease.
Date of Award1 Aug 2017
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorPerdita Barran (Supervisor) & Royston Goodacre (Supervisor)

Keywords

  • MS
  • IM-MS
  • Amyloid-beta
  • alpha-Synuclein

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