Mills, Kevin; (2003) A proteomic approach to the study of inborn errors of metabolism. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This work describes a preliminary study to evaluate the use of proteomics in the study of genetic metabolic disorders. Two metabolic disorders were studied initially, the congenital disorders of glycosylation (CDG) and the α1-antitrypsin deficiencies, both diseases that result in changes in protein expression and post- translational modifications. Strategies were developed using mass spectrometry for the identification and analysis of proteins, in solution and after 2D-PAGE. Using endoglycosidases and proteases in different permutations, methods were developed to monitor the site-specific glycosylation and to increase the amino acid sequence coverage of the pure standard proteins α1-antitrypsin and transferrin. These methods were then applied to proteins purified from patient plasma by affinity chromatography, precipitation or 2D-PAGE. The glycosylation status of the plasma glycoproteins α1-antitrypsin and transferrin was studied in CDG patients with types 1a, 1b, 1c and 1x enzymatic defects. Analysis of the α1-antitrypsin underglycosylated species showed that the glycosylation sites were preferentially occupied in the order 46>247>83. In transferrin glycosylation site 413 was also preferentially occupied to site 611 and site occupancy was not a random process as thought previously. These data having implications for the role of glycosylation and its importance in stabilising the 3D structure of proteins during their maturation, and passage through the quality control system of the cell, prior to their delivery to their site of action. Similarly using 2D- PAGE and mass spectrometry the six major isoforms of α1-antitrypsin were unequivocally identified in control plasma. Using a combination of glycan and peptide analysis, it was possible to confirm the identity of each isoform in the M series of α1-antitrypsin. α1-Antitrypsin was then analysed from 3 patients with known amino acid mutations in the α1-antitrypsin sequence (V213A, E342K and T85M). All three mutations were detected in the α1-antitrypsin expressed in plasma using both 2D-PAGE and MALDI TOP MS. In conclusion, this work describes the preliminary evaluation and the promise of proteomics as a tool in the study of genetic metabolic disease.

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