Title:
Identification And Quantification Of Protein O-GlcNAcylation Using MS-Based Proteomics
Identification And Quantification Of Protein O-GlcNAcylation Using MS-Based Proteomics
Author(s)
Xu, Senhan
Advisor(s)
Wu, Ronghu
Garg, Neha
Garg, Neha
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Abstract
O-GlcNAcylation is a type of protein glycosylation where a single N-acetylglucosamine (GlcNAc) is covalently bound to the serine and threonine residues. It is added by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). O-GlcNAcylated proteins are primarily located in the nucleus and the cytoplasm, but extracellular O-GlcNAcylated proteins have also been reported. This modification has been found to be extensively involved in many cellular processes, including gene transcription, protein translation, and cell cycle controls. As protein O-GlcNAcylation plays a critical role in mammalian cell survival, its dysregulation is associated with many diseases, such as cancer, diabetes, and neurodegenerative diseases.
Despite its importance, O-GlcNAcylated proteins are very difficult to study because of the low abundance of many glycoproteins and the dynamic nature of this modification. Therefore, its global analysis requires effective enrichment of glycoproteins. However, the existing methods have some issues related to the efficiency and specificity. Furthermore, the knowledge of the distributions and functions of many O-GlcNAcylated proteins are not well characterized using conventional biological methods. MS-based proteomics provides a unique opportunity to systematically study protein O-GlcNAcylation at the proteome scale.
My thesis focuses on developing MS-based proteomics method to systematically identify and quantify protein O-GlcNAcylation. In Chapter 2, I have developed a method integrating bioorthogonal chemistry and an enzymatic reaction to simultaneously identify and distinguish glycoproteins modified with O-GlcNAc and O-GalNAc (the Tn antigen), which are two important protein modifications with very similar structures and the same composition, but completely different functions.
In Chapter 3, I have systematically quantified the nuclear-cytoplasmic distributions of O-GlcNAcylated proteins in human cells. The results demonstrate that O-GlcNAcylated proteins with different functions had distinct patterns of the distributions, and the distributions vary varied site-specifically. Moreover, the dynamics of O-GlcNAcylated proteins and non-modified ones in the nucleus and the cytoplasm was comprehensively analyzed, and the half-lives of glycoproteins in these two compartments were found to be markedly different. Additionally, glycoproteins in the nucleus were more dramatically stabilized than those in the cytoplasm under the OGA inhibition.
In Chapter 4, I have comprehensively investigated the nuclear-cytoplasmic distributions of the modified (phosphorylated and O-GlcNAcylated) and non-modified forms of proteins to dissect the correlation between protein distribution and modifications. The results reveal that the different distributions between the modified and non-modified forms of proteins are associated with their functions. Moreover, bioinformatics analyses indicate that other factors are related to the impact of phosphorylation on protein distribution, including protein size and function, local structure, and adjacent amino acid residues around phosphorylation sites.
In Chapter 5, I have developed a novel chemoenzymatic method based on a wild-type galactosyltransferase and uridine diphosphate galactose (UDP-Gal) for global and site-specific analysis of protein O-GlcNAcylation. This method integrates enzymatic reactions and hydrazide chemistry to enrich O-GlcNAcylated peptides. All reagents used are more easily accessible and cost-effective compared with the engineered enzyme and click chemistry reagents.
In Chapter 6, I have developed a quantitative proteomics workflow to systematically determine the stoichiometries of O-GlcNAcylated proteins. The O-GlcNAcylated proteins are enriched by combining metabolic labeling and click chemistry, and the identities of O-GlcNAcylated proteins were confirmed by the site-specific mapping of glycosylation sites. The stoichiometries of glycoproteins are determined by comparing the abundance of the enriched glycoproteins versus the non-enriched flow-through using quantitative multiplexed proteomics.
In Chapter 7, I designed an integrative method to site-specifically identify co-translational O-GlcNAcylation that is of very low abundance in cells but have critical functions in regulating the degradation of nascent polypeptides.
In Chapter 8, I present a chemoproteomics method based on a probe that enables direct detection of the cysteine oxidation sites without the need of prior reduction. The method was first validated by reacting with insulin that contains oxidized cysteines. Next, Using Jurkat cells it was demonstrated that the probe specifically targets oxidized cysteines and does not react with other modifications and amino acid residues. Combining with multiplexed proteomics, we applied the method to study the differences of cysteine oxidation in the livers from mice fed with the high or low fat diet (HFD/HLD). It was found that the cysteine oxidation is dramatically upregulated in the HFD samples, and the upregulation is correlated with protein distribution, functions, and the flanking residues of the oxidation site.
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Date Issued
2022-12-14
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Text
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Dissertation