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

Phillips, Ashley

[Thesis]. Manchester, UK: The University of Manchester; 2016.

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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.