Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.773920
Title: Advanced new technologies for protein analysis by mass spectrometry
Author: Lam, Pui Yiu
ISNI:       0000 0004 7961 1532
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2018
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Abstract:
The work presented herein applies advanced techniques in an ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to study one of the most amyloidogenic amyloid proteins - human islet amyloid polypeptide (hIAPP) and we also demonstrate alternative analytical techniques for proteomics. Exploring the aggregation and inhibition pathways of amyloid proteins is critical for future therapeutic development. Previous studies demonstrated the early, soluble oligomers of amyloid proteins are more toxic to neuro cells than mature amyloid fibrils due to their ability to penetrate and destroy neuro cell membranes. Current analytical techniques for amyloid protein studies, including nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectrometry, transmission electron microscopy (TEM), and atomic force microscopy (AFM), are difficult to perform as they require molecular isolation and focus on studying individual target early oligomers from a mixtures of oligomers. Previous literature, however, has demonstrated the ability of MS in observing as well as isolating a specific oligomer from an in vitro amyloid protein sample. Thus, MS has been applied together with electron-based dissociation technique in this thesis to determine the aggregation as well as inhibition pathways of hIAPP. The results herein demonstrate the region around Gly-33 and Ser-34 of hIAPP is critical for hIAPP aggregation since it is the minimum interaction region between the two hIAPP units which was shown by the electron-based dissociation spectra of the dimer as well as the trimer of hIAPP. hIAPP has also shown to be deamidated rapidly within a month at Asn-21, Asn-22, and Asn-35 residues, regarding there are seven potential deamidation sites within the sequence. The deamidated hIAPP species with iso-aspartic acid residue products at the deamidation sites shows a significant change in the amyloid fibril morphology which may help to explain the previously observed acceleration of aggregation rate by inducing 5% of deamidated hIAPP into non-deamidated hIAPP solution. The methodologies applied in studying the aggregation pathway of hIAPP are further used to explore the inhibition pathways between hIAPP and potential inhibitors. From the results, two different inhibition pathways are proposed to effectively prevent the formation of hIAPP fibrils. Inhibitors interacting specifically at the critical aggregation region around Gly-33 and Ser-34, are shown to prevent the formation of hIAPP fibrils by forming hetero-complexes (a complex composes of hIAPP and inhibitor). The alternative inhibition pathway requires an inhibitor which interacts non-specifically with hIAPP and triggers the formation of non-toxic amorphous aggregates faster than the generation of normal, toxic hIAPP oligomers. These two proposed inhibition pathways show an effective inhibition effect on formation of hIAPP fibrils, however, only the non-specifically interaction pathway was effective in preventing the aggregation of the deamidated from hIAPP since the protein sequence as well as structure are distorted in the deamidated hIAPP which significantly affects the specific interaction between the deamidated hIAPP and inhibitors. The in vitro studies propose the potential aggregation and inhibition pathways for hIAPP; it is critical, but unfortunately very challenging, to study these molecular interactions in vivo due to the solvents and column chemistry required by the current liquid chromatography (LC) approach which is necessary for complex mixture studies. In order to study the in vivo molecular interactions, an alternative analytical technique, therefore, is required. Herein, two dimensional mass spectrometry (2DMS) is applied to study proteomic samples ranging from standard protein digests to whole cell lysate digest without online LC separation. Various sample preparation and 2DMS acquisition methods have been applied to optimise the proteomic outputs from 2DMS. From the results, 2DMS shows a similar capability in studying proteomics compared to the standard LC tandem MS (MS/MS) techniques. The peptides assigned in 2DMS experiments are more hydrophilic, basic, and short, which are complementary to those assigned in the LC MS/MS technique, thus more proteins can be covered by combining the results obtained from 2DMS and LC MS/MS, in which a deeper proteome coverage is achieved. 2DMS is shown to be a viable alternative analytical tool for proteomics, in which is also potential for the in vivo study of molecular interactions in the coming future. The final section of this thesis provides a conclusion on all the works that have been demonstrated herein and discusses the future research which we needed to improve/enhance the current knowledge on amyloid protein studies using MS as well as 2DMS for proteomics.
Supervisor: Not available Sponsor: University of Warwick ; British Federation of Women Graduates ; Great Britain-China Centre ; Sir Richard Stapley Educations Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.773920  DOI: Not available
Keywords: QD Chemistry
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