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Mass spectrometry-based studies of synthetic and natural macromolecules

By Gillian R. Hilton


Originally established as an analytical technique in the fields of physics and\ud chemistry, mass spectrometry has recently become an essential tool in biological\ud research. Advances in ionisation methods and novel types of instrumentation have\ud led to the development of mass spectrometry for the analysis of a wide variety of\ud biological samples. The work presented here describes the use of mass spectrometry\ud to characterise a variety of synthetic and natural macromolecules.\ud Transmissible spongiform encephalopathies (TSEs), also known as prion diseases,\ud are a class of fatal, infectious neurodegenerative diseases that affect both humans\ud and animals. Prion proteins are unprecedented infectious pathogens that cause a\ud group of invariably fatal neurodegenerative diseases by means of an entirely novel\ud mechanism. Ion mobility mass spectrometry (IM-MS) was used to probe the\ud conformation of a variety of different prion proteins in the gas-phase. It was shown\ud that IM-MS could distinguish between two recombinant structures representative of\ud normal cellular prion protein, PrPC and the pathogenic scrapie form (PrPSc). The\ud structure of the full-length prion protein was probed by means of IM-MS. A\ud comparison of the estimated cross-sections of truncated prion protein constructs and\ud full-length constructs suggested that the N-terminal flexible tail was associated with\ud the core structure. Metal binding to two different prion protein constructs was\ud investigated. It was observed that copper coordination to the N-terminal fragment\ud could induce conformational changes in the octarepeat fragment. These changes\ud were relatively small and could not be measured in the full-length prion protein. The\ud data suggested that minor structural changes in the N-terminal could stimulate\ud endocytosis via a minor, undetected, conformational change in the C-terminal\ud domain.\ud IM-MS was used as a high resolution separation technique to distinguish between\ud mixtures of isobaric synthetic polymers. It was observed that the resolving power of\ud IM-MS/MS was insufficient to resolve the higher molecular weight oligomers. In\ud comparison, gel permeation chromatography (GPC)-nuclear magnetic resonance\ud (NMR) spectroscopy (GPC-NMR) analysis of the same isobaric mixture could not\ud separate the two components. It was observed that IM-MS was better than GPCNMR\ud at separating isobaric poly(ethylene glycol) mixtures, especially when taking\ud speed and sensitivity into account

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