Pegg, Hamish;
(2022)
Orthogonal proteomics approaches to discover novel non-haem iron and 2-oxoglutarate dependent dioxygenase catalysed prolyl hydroxylation in the mammalian proteome.
Doctoral thesis (Ph.D), UCL (Univeristy College London).
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Abstract
Oxygen is used in a variety of biochemical processes, including regulation of the transcription factor hypoxia inducible factor (HIFα). Oxygen-sensitive prolyl hydroxylation of HIFα, which is catalysed by the HIFα prolyl 4-hydroxylases (PHDs), enables von Hippel Lindau protein (pVHL) to bind and ubiquitylate HIFα leading to its degradation by the proteasome. The PHDs belong to a large family of non-haem iron- and 2-oxoglutarate-dependent dioxygenases (2OGDs), which includes several other prolyl hydroxylases. Thus, the integration of oxygen into proteins, particularly on proline residues, is a fundamental post-translational modification (PTM). Despite this, the number of known prolyl hydroxylase substrates is relatively small, so orthogonal proteomics approaches using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) were performed to survey the proteome for prolyl hydroxylation in a variety of cellular contexts, including hypoxia, PHD- and pVHL-defective cells, pan-2OGD inhibition, and cells treated with a proteasome inhibitor. Qualitative analysis of hydroxyproline assignments highlighted the confounding role of oxidation occurring on residues in proximity to the reported site of hydroxyproline. With this in mind, the sensitivity of hydroxyproline detection in LC-MS/MS experiments was improved by extensive high pH off-line fractionation and the development of a hydroxyproline affinity purification reagent that enabled endogenous hydroxyproline-containing HIFα peptides to be identified. In total, 49 novel hydroxyproline sites were identified on 34 proteins, including prolyl hydroxylation of 8 protein disulphide isomerases. Further research is required to generate hydroxylase assignments that might provide the framework for functional studies. In addition to identification of novel hydroxyproline sites, oxygen decreased the abundance of 14 proteins and further research is required to determine the mechanism underpinning this apparent decay. Overall, the application of orthogonal LC-MS/MS approaches enabled the identification of many novel hydroxyproline sites and proteins that might be involved in dynamic oxygen-sensing pathways.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Orthogonal proteomics approaches to discover novel non-haem iron and 2-oxoglutarate dependent dioxygenase catalysed prolyl hydroxylation in the mammalian proteome |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| Keywords: | Prolyl hydroxylation, proteomics, SILAC, TMT, VHL, hydroxyproline, LC-MS/MS |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10159336 |
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