Chalkley, Robert James; (2002) Characterisation of 0-GlcNAc Modification Using Mass Spectrometry. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes analytical biochemistry studies in the field of glycobiology, focusing on the use of advanced mass spectrometric techniques as a tool for studying the addition of single N-acetylglucosamine residues to intracellular proteins. Transient glycosylation of nuclear and cytoplasmic proteins by O-GlcNAc has been detected in all eukaryotes studied. This modification is believed to control protein activity, stability and function, and performs a similar, but possibly antagonistic role in controlling the protein to that mediated by phosphorylation. However, a functional role of the modification is only known for a small number of proteins. Consequently, new methods for detecting and characterising the site specificity for this modification will enable more detailed structure/function studies to be carried out, and will provide the basic information required to elucidate the molecular details of biological activity of this post-translational modification. It is the development of these methods that is the subject of this thesis. It is shown that through the application of quadrupole orthogonal-acceleration TOF technology, mass spectrometry can be used to identify sites of O-GlcNAc modification. Firstly, methods are developed for determining sites of modification of O-GlcNAcylated synthetic peptides. Next, these methods are employed to identify a previously known site of GlcNAc modification on ?-crystallin, through analysing solution and in-gel digests of gel purified material. A detailed study of the post-translational state of serum response factor is carried out. In this work previously reported sites of phosphorylation and GlcNAc modification are confirmed and novel sites of O-GlcNAc and phosphate modification are identified. This thesis demonstrates that a mass spectrometric approach is significantly more sensitive than previous techniques employed, and should become an important tool in the development of our understanding of this modification. The background and relevance of this advance in analytical methods, and its potential use in determining the role of O-GlcNAc modification is also discussed.

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