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Chemical proteomics tools for probing carbohydrate processing enzymes

Author: 
Date created: 
2020-04-24
Identifier: 
etd20914
Keywords: 
Chemical proteomics/chemoproteomics
Pull-down
Interactome
Enzyme regulation
Post-translational modification
O-GlcNAc
Abstract: 

The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is a nucleocytoplasmic modification that is present on over a thousand different protein targets. This post-translational modification is conserved among multicellular eukaryotes and has been found to play diverse physiological roles within cells. Remarkably, O- GlcNAc levels are globally controlled by only two enzymes; O-GlcNAcase (OGA) and O- GlcNAc transferase (OGT). How this modification is regulated on the large set of target substrates by just these two enzymes remains a topic of high interest within the field. In this thesis, I describe the development of a chemical proteomics method to interrogate OGA that help move the field toward elucidating the factors that regulate this enzyme. Specifically, I describe the creation of four distinct affinity-based probes that bind with high affinity to OGA and enable its precipitation at endogenous levels from tissues. These probes are designed to enable precipitation of OGA and its protein partners under gentle conditions followed by precipitation on streptavidin-coated beads. The disulfide linker that can be cleaved using gentle conditions enables release of OGA-containing protein complexes. Using these probes in a parallel series of experiments, I define a set of high confidence candidate OGA interacting proteins that are seen in multiple data sets from mass spectrometry-based proteomic analyses of the chemoproteomic precipitates obtained from bovine brain tissue. In addition, I detail targeted discovery of post- translational modifications on OGA from bovine brain tissue obtained using one of these new chemoproteomic probes. I envision this approach can ultimately be applied to identifying factors that regulate OGA activity within cells and provide a blueprint for robust chemoproteomics strategies that harness the use of multiple probes having distinct chemical structures.

Document type: 
Thesis
Rights: 
This thesis may be printed or downloaded for non-commercial research and scholarly purposes. Copyright remains with the author.
File(s): 
Supervisor(s): 
David Vocadlo
Leonard Foster
Department: 
Science: Department of Chemistry
Thesis type: 
(Thesis) M.Sc.
Statistics: