This thesis describes the development and evaluation of a new laboratory tool to improve early kidney injury diagnosis. In the first part, we define the unmet clinical needs in the clinical care... Show moreThis thesis describes the development and evaluation of a new laboratory tool to improve early kidney injury diagnosis. In the first part, we define the unmet clinical needs in the clinical care pathways in kidney injury, by a questionnaire and s literature review to summarize current state-of-the-art diagnostics. The second part of this thesis comprises the analytical phase of test development. Liquid-chromatography (LC) coupled to tandem MS was chosen as analytical platform for protein-based kidney injury biomarker quantitation from human urine specimens. The third part of this thesis covers aspects of the postanalytical phase of medical test development, which included the establishment of reference intervals and clinical observational cohort studies. Elevated levels of the studied kidney injury biomarkers, and their combined signatures, were associated with acute kidney injury induced by ischemia-reperfusion injury after kidney transplantation, but not with stable chronic kidney disease. In a general perspective, this thesis highlights the role of quantitative protein mass spectrometry for biomarker translation from research towards the clinical laboratory. Show less
The research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating... Show moreThe research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating enzymes. These enzymes are responsible for the conjugation and the removal of the post-translational modifier ubiquitin. This small protein is involved in almost all cellular processes, and when conjugated onto a substrate protein it can signal for degradation and influence the localization, interaction, stability and activity of the protein. Therefore, the dysregulation of these processes can have detrimental effects of cell organization and survival which in turn has implications in numerous processes related to diseases. Hence, it is important to fully understand the ubiquitination pathway and how to interact with it. The ubiquitin-based research tools described in this thesis aim to shine light on parts of this pathway. Ranging from the selectivity and specificity of DUBs for specific Ub linkages in competition and the catalytic efficiency of these proteolytic cleavage processes to the selectivity and activity of ligases and the activity of DUBs in cells. All ubiquitin research tools are based on synthetic ubiquitin modified with unnatural amino acids, neutron-encoded amino acids, point mutations and/or fluorescent labels, in order to study the characteristics of the enzymes in vitro. Show less
Glycosylation is a widely occurring and complex modification found on lipids and proteins and is involved in the recognition, signaling and interaction events within the cell and between cells.... Show moreGlycosylation is a widely occurring and complex modification found on lipids and proteins and is involved in the recognition, signaling and interaction events within the cell and between cells. These events based on glycan structures result in adhesion, cell-matrix interaction and immune recognition. Alterations in the glycomic profile are considered a hallmark of various diseases, including cancer where it contributes to the development and progression of cancer, affecting cell-cell communication, cell-matrix interactions, tumor angiogenesis, invasion and metastasis. These functions are governed by different glycans and their terminal structures. In order to further explore these structures with regard to their potential as biomarkers and specific targets for diagnostic applications and therapeutical strategies for various diseases, in-depth glycomic analysis is needed. It is further noted that aberrant glycosylation not only results from the altered expression of glycosyltransferases (GTs) but also from the changed activity of GTs and glycosidases as well as the availability and abundance of sugar nucleotide donors. The aim of the research described in this thesis was to explore the glycomic signatures of colorectal cancer (CRC) in cell lines and tissues as well as of acute myeloid leukemia (AML) cell lines. Show less
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
Biomarker molecules are analyzed in clinical tests to diagnose a disease, but often these test lack sensitivity or specificity. Also, for many diseases there is not even a blood based test... Show moreBiomarker molecules are analyzed in clinical tests to diagnose a disease, but often these test lack sensitivity or specificity. Also, for many diseases there is not even a blood based test available, while blood collection is relatively low invasive. For breast- and pancreatic cancer, there are several proteins that could potentially serve as biomarkers in blood, but these are not yet specific enough to use for diagnostic testing. Further research on other types of biomarkers may therefore be a valuable addition to eventually be able to develop a blood test. Methods for glycosylation profiling from serum and dried bloodspots with mass spectrometry were developed and applied to pancreatic- and breast cancer biomarker studies. Differences were found between profiles of healthy and sick persons for pancreatic cancer, but no clear differences were seen for breast cancer. This is probably due to the many different forms of breast cancer which result in different profiles. In the future, combining different types of markers from serum might ensure that differences between healthy and sick, between different diseases and between types of disease can be identified. This could lead to the development of a blood test for the early detection of cancer and other diseases. Show less
The thesis describes the development of a number of novel mass spectrometric methods for the protein analysis of Gram-negative bacteria. These applications are developed with the aim of finding new... Show moreThe thesis describes the development of a number of novel mass spectrometric methods for the protein analysis of Gram-negative bacteria. These applications are developed with the aim of finding new and improved diagnostic routes for the typing of bacteria and their antibiotic resistance. The research is application driven and the focus is on utilizing high-end mass spectrometric instrumentation in diagnostic clinical microbiology, in a complimentary nature to already established techniques. Show less
Glycosylation is an important way in which proteins, the functional agents of our body, can be modified to alter and expand their functional repertoire. Glycans consist of monosaccharides that... Show moreGlycosylation is an important way in which proteins, the functional agents of our body, can be modified to alter and expand their functional repertoire. Glycans consist of monosaccharides that are linked in a chained and branching fashion, often to form specific epitopes that are of clinical and biopharmaceutical interest. In order to study glycosylation, there is a need for high-throughput analysis methodology. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a prominent example of this, as it can rapidly provide information on the monosaccharide compositions of glycans. However, it is challenging for the method to yield information on the structural aspects of glycosylation, as well as on glycans carrying sialic acids. These sialylated glycans are prone to in-source and metastable decay, and tend to require chemical derivatization to allow their analysis. The aim of this thesis is the development and application of new methodology for MALDI-MS N-glycomics, and, with a focus on metabolic syndrome and rheumatoid arthritis, to increase our understanding of the role of N-glycosylation in health and disease. A principal outcome of the work is a sialic acid derivatization protocol that allows the mass-based discrimination of alpha-2,3- and alpha-2,6-linked sialic acids, facilitating their study in a high-throughput setting. Show less
The capability of cells to divide is essential for all organisms, while uncontrolled cell proliferation can have detrimental effects resulting in diseases like cancer. Cell division is... Show moreThe capability of cells to divide is essential for all organisms, while uncontrolled cell proliferation can have detrimental effects resulting in diseases like cancer. Cell division is therefore tightly controlled by regulatory mechanisms. Post-translational modifications (PTMs) are able to directly change the function of a protein and thereby provide a quick functional switch. This thesis focusses on the roles of small ubiquitin-like modifiers (SUMOs) and their crosstalk with other post-translational modifications during cell division, at the proteome-wide level as well as the single target protein level. Show less
This dissertation describes the development of glyco-bioinformatics tools that facilitate the high-throughput data processing of glycomics and glycoproteomics experiments, specifically for both... Show moreThis dissertation describes the development of glyco-bioinformatics tools that facilitate the high-throughput data processing of glycomics and glycoproteomics experiments, specifically for both MALDI-TOF-MS (Chapter 2) and LC-ESI-MS (Chapter 3). The developed methods also provide various quality control parameters that assist the researcher in curating both the measured spectra and quantified analytes, thereby providing high-quality data in a high-throughput manner.The tools that were developed within this thesis have been used to identify the influence of glycosylation on trypsin efficacy of Immunoglobulin G (Chapter 3) and two biological cohorts. Specifically, to investigate the serum N-glycosylation during and after pregnancy (Chapter 5) and to identify the differences in the N-glycosylation between maternal and fetal serum and IgG (Chapter 6). Show less
Glycosylation of immunoglobulins is suspected to play a key role in the regulation of the immune system. In this thesis, mass spectrometry-based glycoproteomics methods were used to characterize... Show moreGlycosylation of immunoglobulins is suspected to play a key role in the regulation of the immune system. In this thesis, mass spectrometry-based glycoproteomics methods were used to characterize the glycosylation of various immunoglobulins. In Chapter 2 we describe the development of a glycoproteomics method to analyze IgE glycosylation. In Chapter 3 we reported partial O-glycosylation of IgG3. In addition to structural glycosylation research, we also analyzed antibody glycosylation in population cohorts. In Chapter 4 and Chapter 5, IgG Fc glycopeptide analysis was performed on blood samples using LC-MS(/MS). In a cohort of 76 ANCA vasculitis patients, low galactosylation and sialylation of IgG was associated with a higher chance of future relapse. Furthermore, in the approximately 1800 participants of the Leiden Longevity study (LLS), low galactosylation and sialylation of IgG, together with high fucosylation, showed association with markers of inflammation. We hope that the novel data presented in this thesis may contribute to the elucidation of the role of antibody glycosylation in the immune system, of which the understanding is currently still very limited. Show less
Glycan modifications of proteins and lipids form an integral part of the cell’s outermost layer and an array of ligands, adding a high degree of complexity to the cellular phenotype. While... Show more Glycan modifications of proteins and lipids form an integral part of the cell’s outermost layer and an array of ligands, adding a high degree of complexity to the cellular phenotype. While this complexity is an analytical challenge, it also offers a wide range of opportunities for biomarkers and treatment targets. This thesis deals with the analysis of colorectal cancer (CRC)-associated glycomic changes. Current knowledge on CRC-associated glycan changes and their biological role have been reviewed in Chapter 1. In Chapters 2, 3, and 6, we developed novel, high-end methodologies for the glycomic analysis of tissues and cell lines to be able to expand our knowledge on cancer glycomics and to overcome some limitations of current techniques. By applying these new methods, this thesis also covers the characterization of changes in glycosylation in CRC tissues as well as cell lines, thereby contributing to the understanding of CRC biology while identifying cancer-specific signatures underlying CRC development. These signatures can be further explored as potential markers to improve patient care. Additionally, in Chapter 5, we extended our research to pancreatic duct adenocarcinoma and characterized the N-glycome of PDAC cells with different metastatic potential and of a normal pancreatic duct cell line. Show less
