The 2-oxoglutarate and ferrous ion dependent oxygenases are a super family of enzymes that are involved in a wide range of biological processes regulated trough the mechanism of post-translational modification (PTM). Such biological processes include hypoxia sensing (through regulating HIF transcription), fatty acid metabolism (through carnitine production), transcriptional regulation (through demethylation of histones), and collagen structure formation (through proline and lysine hydroxylation). To understand the underlying mechanisms of such regulatory processes, and to develop clinically useful inhibitors, and thereby regulate these processes in living organisms, requires sensitive methods for monitoring enzyme activity. The use of indirect methods such as quantification of reaction products (14CO2 or succinate) can be problematic, as both products can result from competitive reactions. Alternative direct measurement of substrate modifications using mass spectrometry-based proteomics can be applied; however, (1) for this technique the limit of detection is often prohibitive, (2) the method is best suited for the confirmation of known modifications, rather than for the discovery of new modifications, and (3) sequence coverage may often be only 60%, and therefore many modifications can be missed. The aim of the research presented in this thesis was to develop amino acid analysis and to apply these methods to the identification and quantification of PTMs catalysed by 2-oxoglutarate and ferrous ion dependent oxygenases. A range of LC-MS approaches were investigated including: (1) C18 reversed phase chromatography of quinoline derivatised amino acids, (2) ion paring chromatography, and (3) mixed mode chromatography with either UV, or conventional molecular MS, or isotope ratio mass spectrometry detection. Analysis of the elution patterns for those separation techniques enabled estimation of the retention parameters of modified amino acids and the identification of the modifications, where no standards were available. The most sensitive approach developed employed mixed mode chromatography coupled to isotope ratio mass spectrometry which was optimised for the analysis of modified amino acids. This was shown to have a limit of detection two orders of magnitude lower (0.01μM) than other conventional mass spectrometry techniques. Using amino acid labelling in cell culture, a quantification protocol was developed which employed a non-labelled internal standard and selectively labelled cell culture. The method was shown to be suitable for both very accurate quantification at low concentration levels and metabolic studies, allowing us to track back the modifications to their precursors. The analytical methods developed for amino acid analysis were successfully applied to the analysis of modifications resulting from 2-oxoglutarate and Fe dependent oxygenase activity. Stereochemistry of lysyl hydroxylation in the splicing regulatory protein Luk7L2 by JmjD6 as well as of the self hydroxylation of the JmjD6 was identified. The stereochemistry was shown to be different from that of previously reported for the collagen hydroxyline, hydroxylated by the another member of this enzyme family. mbP4H enzyme was shown to catalyse prolyl hydroxylation of taODD resulting in 4R-hydroxyprolyl. Amino acid analysis was used in order to verify the mechanism of the hBBOX catalysed rearrangement of Mildronate. Using the method developed for the analysis of non-derivatised amino acids the screening of potential substrates of hBBOX enzyme was carried out and two new substrates were identified. The isotope ratio mass spectrometry protocol was applied to the study of histones from cell culture; low levels of hydroxylated and methylated amino acids were quantified. The analytical methods described were developed to complement to the well established proteomics techniques. The methods developed enable investigation into the region- and stereo- chemistry of the modified groups within the modified AA residue and has proved to be a powerful tool of exploratory PTM analysis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:604506 |
| Date | January 2014 |
| Creators | Loik, Nikita D. |
| Contributors | McCullagh, James S. O. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:8c737f3c-b24d-437d-abac-bada61351173 |
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