A Wholistic “Omics” Approach to Understanding the Biological Roles of Glycans in Biological Systems

Date

2023-05

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Abstract

Proteins are complex molecules with direct biofunctions in cells, tissues, or organisms. After the translation of proteins, some amino acid residues are usually modified by post-translation modifications (PTMs). One of the common PTMs is known as glycosylation, which is a key process to covalently attach glycans to proteins and ensure the proper folding and stability of proteins. The glycan moieties of glycosylated proteins provide various functions, such as intercellular signaling and immune response. Aberrant glycosylation has been reported in several diseases, therefore, certain glycans or glycopeptides can be recognized as potential biomarkers for prognosis or diagnosis. The study of glycomics involves characterization and quantification of glycans derived from biological samples. The common process of sample preparation includes the release of glycans from proteins, followed by purification. On the other hand, glycoproteomics focuses on glycopeptides, which profiles both glycan structure and peptide sequence. Therefore, information of glycosylation sites can be acquired. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a powerful tool for identification of biomolecules due to its high resolution and sensitivity. It has been widely used in both glycomics and glycoproteomics analyses. However, glycosylation studies remain challenging because of the low ionization efficiency of hydrophilic glycans, microheterogeneity of isomeric glycan structures and low abundance of glycopeptides. Therefore, this dissertation includes the development of novel methods to combat the above challenges. In this dissertation, the glycome and proteome profiling of cancer cell lines using LC-MS/MS methods are included. A recently developed physiological culturing medium called Plasmax has been reported with the nutrients and metabolites that are normally found in human plasma. To compare this novel medium with commercially available media, one breast cancer cell line and one brain cancer cell line were cultivated in Plasmax and other two commercial media DMEM and EMEM. Glycome profiling indicates that galactosylation and branching of N-glycans were significantly affected by Plasmax. Moreover, the lowest abundance of O-glycans was observed in EMEM, suggesting that EMEM is not suitable for cell line-based O-glycomics studies. The expression changes of glycans were also correlated with the expressions of glycogenes of the enzymes that are responsible for the synthesis of glycans. On the other hand, bottom-up proteomics analysis was also performed to investigate the protein expression changes, which resulted in alterations of cellular functions, such as invasion and migration. Together, the glycomics and proteomics studies aim to provide comprehensive understandings of Plasmax and aid media selection for future studies of cell lines. Another major part of this dissertation is method development. An extra-long reversed-phase liquid chromatography (RPLC) C18 column was utilized under high temperature to separate permethylated glycan isomers. Permethylation has been recognized as a reliable method to improve the structural stability and ionization efficiency of glycans. Also, the higher hydrophobicity of permethylated glycans allows them to achieve better separation using RPLC. In this work, the separation of both sialylated linkage isomers and fucosylated positional isomers were achieved. In addition, hydrophobicity factor of glycopeptide was introduced to normalize the retention time of glycopeptides analyzed by different LC-MS/MS systems. HILIC method was applied for glycopeptide enrichment to reduce the competitive ionization using MS analysis. A library of identified human serum glycopeptides with corresponding hydrophobicity factors was generated. Another method using acetone precipitation for glycan purification is also investigated. The oxidative release of glycans from egg yolks was achieved using sodium hypochlorite (NaClO), then purified using acetone precipitation to remove proteins and peptides. Unlike the traditional approach, this method does not involve enzymes or C18 cartridges. Therefore, it has the potential to be applied for large scale production of glycans from natural products.

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Keywords

Glycomics,, Proteomics,, LC-MS/MS, Permethylation, Glycorpteomics

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