01whole.pdf (24.12 MB)
Download file

Optimisation of glycomics analysis and the effect of inflammation on protein glycosylation in the central nervous system

Download (24.12 MB)
thesis
posted on 29.03.2022, 02:52 authored by Christopher David Ashwood
Glycomics aims to comprehensively study the entire complement of sugars found in an organism and ideally needs to detect and quantify each individual glycan structure present. Using liquid chromatography in tandem with mass-spectrometry allows separation, characterisation and quantitation of released glycan structures from biological samples, improving our understanding of the role glycosylation plays in life. For the nervous system, which is the most essential signalling system in the human body, the role of protein glycosylation is still largely not understood. The application of liquid chromatography with mass-spectrometry for glycan analysis has been frequently performed over the past twenty years but the analysis of generated data is largely manual. Furthermore, there is no established workflow for the quantitation on glycans, independent of instrument manufacturer. Addressing this gap, we have established or optimised methods for obtaining glycan data from LC-MS systems, improved the characterisation of glycans using reproducible fragmentation methods and retention time normalisation and automated the assignment of glycan structure in challenging applications. Toll-like receptor 4 is one of the research targets for understanding the signalling pathways involved in the human pain response due to its role as a membrane protein which mediates inflammation to both foreign and endogenous molecules. As glycosylation plays a role in cell-to-cell signalling, it is expected, but not yet established, that the activation of the toll-like receptor 4 pathway will result in changes to the cell surfaces involved in transmitting the signals involved in pain transduction. Furthermore, the abundance of toll-like receptor 4 in normal and activated signalling states has not yet been understood, despite the importance of receptor abundance in understanding a signalling pathway. We have approached this signalling pathway with both in vivo and in vitro models observing up-regulation of core-fucosylation on proteins secreted by cell lines and identified a positive correlation of the paucimannose glycan class on specific brain regions to increased pain sensitivity. In addition, we have described the detection of toll-like receptor 4 in published studies and developed targeted methods to detect it and its partner, myeloid-differentiation factor 2.

History

Table of Contents

Chapter 1. Introduction -- Chapter 2. Materials and methods -- Chapter 3. Optimisation of LC-ESI-MS parameters for quantitative glycomics -- Chapter 4. - Improving glycan characterisation by fragmentation -- Chapter 5. Automating glycan isomer discrimination using diagnostic ions -- Chapter 6. Improving glycan characterisation by normalised retention time on Porous Graphitised Carbon Chromatography -- Chapter 7. Characterising Protein N-glycosylation changes following TLR4 activation -- Chapter 8. Detection of the TLR4 protein complex by mass spectrometry -- Chapter 9. Summary and future directions -- References -- Appendices.

Notes

Glyco@MQ; Department of Molecular Sciences, Macquarie University, Sydney, Australia" -- title page. Bibliography: pages 187-214 "ARC Centre of Nanoscale Biophotonics Thesis by publication.

Awarding Institution

Macquarie University

Degree Type

Thesis PhD

Degree

PhD, Macquarie University, Faculty of Science and Engineering, Department of Molecular Sciences

Department, Centre or School

Department of Molecular Sciences

Year of Award

2018

Principal Supervisor

Nicolle Packer

Additional Supervisor 1

Mark Molloy

Rights

Copyright Christopher David Ashwood 2018. Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (ix, 242 pages) diagrams, graphs, tables

Former Identifiers

mq:71991 http://hdl.handle.net/1959.14/1280393