Analytical assay development, particularly pertaining to glycomics, is an exciting amalgam of biology, chemistry and engineering. Besides academic research in natural and medical sciences,... Show moreAnalytical assay development, particularly pertaining to glycomics, is an exciting amalgam of biology, chemistry and engineering. Besides academic research in natural and medical sciences, glycomics assays have immense importance in industrial applications such as in quality control and quality assurance of glycoproteins. An up-coming industrial and clinical application is the high-throughput glycan profiling of clinical samples, such as plasma, for identifying disease associations. These glycomics assays are often based on chromatographic and mass spectrometric instrumentation. Thus, they create a requirement of instrumentation infrastructure as well as technical skills which are both not always readily available. This creates a demand in industry for the development of glycomics assays that have a low infrastructure cost as well as minimal training requirements and that are user-friendly. With these objectives in focus, this thesis develops novel exoglycosidase-based high-throughput glycomics assays for use in industrial glycan profiling. In doing so, this thesis also contributes to the development of potential products, such as glycomics kits. Show less
Immunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19.... Show moreImmunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19. Importantly, effector functions and resulting inflammatory responses are impacted by the structure of N-glycans linked to the Fc-tail of IgG. Studying antibody glycosylation in emerging infectious diseases such as SARS-CoV-2 allows to gain insight into specific glycan signatures at the early stages of infection, and to investigate whether these reflect how the disease would progress. For example, low fucosylation is a common glyco-phenotypic signature of IgG1 produced against the spike (S) protein of severely ill SARS-CoV-2 infected patients early on in their disease course, but has likewise been described in other disease settings, where the antigen is presented in the context of host-cell membranes (Chapter 2). In this thesis, antibody glycomics signatures of SARS-CoV-2 infection and vaccination have been explored using an established liquid chromatography – mass spectrometry-based method relying on affinity-isolation and proteolytic digestion of both total and anti-S IgG. In Chapter 3, the glycosylation of SARS-CoV-2 anti-S IgG antibodies were found to be vastly skewed relative to total IgG and to change in a highly dynamic fashion. Moreover, IgG glycosylation was shown to be an early severity marker and showed patient stratification potential, with predicting power for intensive care admission within a hospitalized patient population. Early detection of a pro-inflammatory glycosylation pattern may provide a broader intervention window and decrease the number of ICU-admissions. Furthermore, anti-S IgG1 glycosylation levels obtained with LC-MS show promise to supplement clinical parameters and biomarkers of inflammation, that have together been used for the severity score calculation of hospitalized COVID-19 patients. Similarly to SARS-CoV-2 infection, antibodies generated against the spike protein upon BNT162b2 mRNA vaccination also induced a transient afucosylated anti-S IgG1 response in antigen naïve individuals, albeit to a lower extent than in severely ill patients, exemplifying the influence of the type of immunization on antibody glycosylation (Chapter 4). Upon vaccination, the observed initial, mild afucosylated response was additionally accompanied by low fucosyltransferase (FUT8) expression in antigen-specific plasma cells. Furthermore, the observed initial anti-S IgG afucosylation signature may aided mounting a stronger immune response, as indicated by its correlation with antibody amounts following the second vaccination dose. Given the impact of glycosylation on antibody function, deciphering theunderlying regulatory mechanisms influencing IgG glycosylation will be of great importance to better understand the inflammatory potential, vaccine efficacy and protective capacity of vaccine- or pathogen-induced IgG in both body fluids and tissues in the future.In Chapter 5 and 6, the reaction steps of a previously developed linkage-specific sialic acid derivatization workflow were studied in more detail. Key players in such reactions are catalyst, of which novel types with different physico-chemical properties were introduced in Chapter 5. In Chapter 6, prior lactone formation was found to be a prerequisite for subsequent amidation of α2,3-linked sialic acids, which proceeds via direct aminolysis of the C2 lactone. Together, these new insights will be beneficial for the rational optimization of high-throughput (MALDI-)MS-based glycomics and glycoproteomics workflows relying on linkage-specific sialic acid derivatization. Show less
The surface of eukaryotic cells contains a very dense layer of oligosaccharides called glycans that are linked to protein and lipid carriers and play an important role in cell-cell and cell... Show moreThe surface of eukaryotic cells contains a very dense layer of oligosaccharides called glycans that are linked to protein and lipid carriers and play an important role in cell-cell and cell-extracellular matrix interactions. Cancer-induced changes in glycosylation have an impact on the function of major glycoproteins in the human colon, therefore studies focused on colorectal cancer (CRC)-specific glycosylation signatures can provide novel insights into onset and progression of this disease. The major focus of this thesis was to investigate mucin type O-glycosylation signatures of CRC. For this purpose, a protocol for in-depth analysis of N- and O-glycans obtained from cell lines was developed (Chapter 2) using nanoscale porous graphitized carbon liquid chromatography coupled to mass spectrometry (PGC-nano-LC-MS). In Chapter 3 additional conditions were optimized in the MS methodology by using polar protic dopant (methanol and isopropanol) enriched nitrogen gas to increase sensitivity on the MS and tandem MS level. In Chapter 4 we applied the methodology developed in Chapter 2 to the analysis of O-glycosylation signatures of 26 different CRC cell lines. This analysis resulted in the characterization of more than 150 O-glycan structures and increased our understanding of glycan expression in the analyzed cell lines. To gain further understanding in the mechanisms underlying glycomic changes with colon cell differentiation, we explored changes in the cell line glycome and proteome upon spontaneous and butyrate-stimulated differentiation in in vitro cell culture (Chapter 5). By performing an integrative approach, we generated hypotheses about glycosylation signatures of specific cell adhesion proteins, which may play an important role in cancer progression. The localization of glycans on the cell surface and their role in biological processes are important in cancer pathogenesis, making them potential candidates for glycan targeting immunotherapy. Therefore, we further optimized the methodology to enable comprehensive analysis of N- and O-glycans from specific regions of formalin-fixed, paraffin-embedded tissues using laser capture microdissections and applied it for the analysis of selected regions of CRC tissues and their patient-matched colon mucosa controls (Chapter 6). We identified specific tumor-associated carbohydrate antigens (TACAs) that show expression only in the tumor samples, with no or limited expression in the normal colon mucosa. Since TACAs are present in high abundance on the surface of cancer cells which are linked to many different proteins, these are very promising targets for the development of tumor-specific immunotherapy. Show less
