|Institution:||University of Leeds|
|Full text PDF:||http://etheses.whiterose.ac.uk/11887/|
The study of protein/peptide folding, misfolding, structure, and interactions are vital to understanding complex biological problems. The work presented in this thesis describes the development and application of mass spectrometry -based techniques to investigate protein structure, aggregation and interference with aggregation, providing insights into the self-assembly and inhibition of several disease-related peptides. Mass spectrometry has evolved significantly over the past decade, its applications varying from small molecules to macromolecules. Travelling wave ion mobility spectrometry (TWIMS), when combined with mass spectrometry (MS), has the unique and unrivalled capability of separating molecular ions based on their collision cross-sectional area in addition to their mass-to-charge ratio, thus facilitating structural studies of co-populated protein conformations and structural isomers of protein complexes that cannot be separated by molecular weight alone. One biological system that has benefitted enormously from such advances is the study of in vitro amyloid formation. The ability of amyloidogenic protein/peptides to assemble into insoluble fibrils is the basis of a vast array of human disorders. Human islet amyloid polypeptide (hIAPP) is one such peptide able to readily assemble into amyloid fibrils in vitro at neutral pH, despite being intrinsically disordered. Identifying oligomeric states occupied between monomer and final fibrils creates an enormous challenge, given that few techniques are able to separate and characterise such lowly-populated and transient species. In addition to characterisation of fibril precursors, recent research has focussed on the identification of small molecule inhibitors of the amyloid cascade and understanding their mechanism of action is of great interest to this field. In the work presented here, the power of TWIMS-MS has been harnessed to achieve the separation and characterisation of oligomeric precursors of the type-2 diabetes-related peptide hIAPP along with IAPP mutants and peptides corresponding to its core sequence. In addition, the effects of small molecule inhibitors on oligomer population and fibril formation have been studied and described in detail. Further, an experimentally simple, in vitro MS-based screen has been developed and implemented that provides rapid and accurate analysis of protein aggregation and its inhibition. All of the results highlight the powers of MS to provide important insights into the mechanism of amyloid formation and demonstrate the potential of this approach for screening for novel inhibitors of disease-related amyloid assembly.