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Title: Development and application of proteomic approaches for the study of post-translational modifications
Author: Phillips, Helen Louise
ISNI:       0000 0004 2740 2130
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2012
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Post-translational modifications (PTMs) increase the functional diversity of the proteome by the addition or removal of chemical groups. PTMs determine the way a protein functions, from the way it folds to produce its three-dimensional conformation, and its localisation, to its interactions with other proteins and nucleic acids. Therefore, identifying the nature and sites of PTMs is crucial to the study of cell biology and the pathology of diseases. This Thesis presents the development and application of proteomic approaches to study PTMs in a number of biological systems. PTMs are often difficult to study due to their low abundance within a complex sample. Therefore, the development and application of novel approaches to assist in the identification of PTMs will improve our understanding of their role within the proteome. A novel two dimensional peptide separation technique using mixed mode (reversed phase- anion exchange) chromatography in the first dimension in conjunction with RP-LC-MS/MS was developed. This demonstrated improved efficiency of separation, resulting in improved proteome coverage compared to conventional SCX. The mixed-mode separation technique was subsequently used in the shotgun proteomic analysis of the thermophilic archaeon, Sulfolobus solfataricus. An extensive methylproteome was identified, and suggested to play a role in the thermal stability of the S. solfataricus proteome. Mixed-mode chromatography was also utilised in the shotgun proteomic analysis of synovial fluid from a Rheumatoid Arthritis patient. Two sites of protein citrullination were identified, including a previously unidentified site. In addition, proteomic approaches were applied to the study of a mammalian multifunctional nuclear protein, PSF. The effects of arginine methylation on localisation, RNA-binding, and its interaction with p54 were studied. Furthermore, proteomic approaches were utilised to study a novel putative toxin, BPSL1549 from Burkholderia pseudomallei. Burkholderia pseudomallei is the causative agent for melioidosis, a disease that causes pneumonia and septicaemia and is endemic in southeast Asia and northern Australia. Following demonstration of the cytotoxic effects of BPSL1549 by incubation with eukaryotic cells and lethal administration to mice, mass spectrometry was used to identify the substrate for the toxin as eukaryotic initiation factor 4A. Moreover, MS studies were used to demonstrate BPSL 1549 is a glutamine de amidase and is responsible for deamidation of Gln339 to Glu in eukaryotic initiation factor 4A. BPSL1549 is the first cytotoxin to be confirmed in B. pseudomallei.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available