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 chemical and structural heterogeneity of toxoid vaccines makes their analysis challenging. However, detailed insights on a molecular level can be obtained by mass spectrometry. Our initial... Show moreThe chemical and structural heterogeneity of toxoid vaccines makes their analysis challenging. However, detailed insights on a molecular level can be obtained by mass spectrometry. Our initial focus was the identification of formaldehyde-induced modifications in diphtheria toxin, which is described in Chapter 2. Subsequently, the methods described in Chapter 2 were applied to study what effects formaldehyde-induced modifications on model proteins have on their susceptibility to enzymatic proteolysis (Chapter 3). During the analysis of these model proteins, unknown formaldehyde-induced modifications were observed. The structural elucidation of these modifications, the discovery of a new type of crosslinks and various other subsequent reaction products are described in Chapter 4. The degradomics analysis described in Chapter 3 was applied to tetanus toxoids to distinguish heat-denaturated toxoids from their original state (Chapter 5). In order to reduce the analysis time and further improve the degradomics approach, an optimized strategy using Tandem Mass Tag multiplexing for the relative quantification of peptides was developed for the analysis of diphtheria toxoids (Chapter 6). Finally, Chapter 7 provides a brief discussion on the results presented in this thesis and offers some perspectives on implementation of the findings for toxoid vaccine development, quality control and further research. Show less
The challenge of achieving fast quantification in metabolomics is the presence of severe matrix effects during the MS analysis of complex samples. Complex samples also result in challenges during... Show moreThe challenge of achieving fast quantification in metabolomics is the presence of severe matrix effects during the MS analysis of complex samples. Complex samples also result in challenges during metabolite identification as complex MS/MS spectra and peak overlap in 1H NMR complicate structure elucidation. The goal of this thesis is to tackle these challenges by the development and application of innovative fractionation approaches and state-of-the-art MS and NMR analyses. 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
Advanced mass spectrometry of glycosphingolipids takes the central stage in this thesis. Investigations focus on characterization of glycosphingolipid metabolism in health and disease with... Show moreAdvanced mass spectrometry of glycosphingolipids takes the central stage in this thesis. Investigations focus on characterization of glycosphingolipid metabolism in health and disease with emphasis to the detection and accurate quantitation of known and so far unknown glycosphingolipids and closely regulated metabolites. Inherited defects in lysosomal degradation of glycosphingolipids, in particular the glycosphingolipidoses Gaucher disease (GD) and Fabry disease (FD), relatively common lysosomal storage disorders, are key topics of examination. The thesis provides an introductory background on the field of research and contains three different sections describing conducted experimental work. Section one consists of studies reporting on the discovery of excessive occurrence of glycosphingoid bases in lysosomal storage diseases, the development of methods for their accurate quantitation in biological samples with UPLC-ESI-MS/MS and the use of these methods in diagnosis and disease monitoring. The great value of identical 13C-encoded (glyco)sphingolipids and their bases as internal standards in mass spectrometric quantitation of these lipids in biological materials is described. Section two introduces clinical aspects and challenges of GD and FD and provides examples of the practical value of lipid analyses in the GD and FD clinic. Section three concerns the pathophysiology of lysosomal disorders in glycosphingolipid metabolism and related fundamental investigations. 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
In this thesis, metabolomics is used to study the role of the host-virus interaction on a metabolic level. A special emphasis is directed on the role of inflammation and oxidative stress on... Show more In this thesis, metabolomics is used to study the role of the host-virus interaction on a metabolic level. A special emphasis is directed on the role of inflammation and oxidative stress on the metabolic level, as part of the innate immune response against viral infection. We chose respiratory syncytial virus (RSV) and hepatitis B virus (HBV) as candidate viruses to metabolically study their role in acute respiratory infection and chronic hepatitis B infection. Secondly we also investigated infant metabolic and immunological consequences of in utero exposure to antiretroviral intervention and human immunodeficiency virus (HIV). Collectively, established targeted metabolomics approaches in conjunction with newly developed metabolomics methodologies and complemented with other “omics” techniques, were used to address pertinent questions related to host metabolic functioning and alterations during viral infection. In vitro RSV studies together with in vivo patient based studies relating to chronic HBV infection and in utero exposure too antiretroviral and HIV were used to address these questions. The work is divided into three research parts containing: i. the analytical methodology development work, ii. in vitro based metabolomics and iii. patient based metabolomics. Show less
The main theme of this thesis, allosteric modulation effectuated through the sodium ion site of GPCRs, is inspired by the important role that this site appears to play in GPCR signaling. As... Show moreThe main theme of this thesis, allosteric modulation effectuated through the sodium ion site of GPCRs, is inspired by the important role that this site appears to play in GPCR signaling. As sodium ions are abundant under physiological conditions they may affect GPCR signaling considerably. Receptor activation causes a substantial rearrangement of the sodium ion site, suggesting an important role in this process. Chapter 2 reviews the current knowledge on allosteric modulation of amiloride and its derivatives binding to the sodium ion site of Class A GPCRs. Chapters 3 to 5 follow-up on the recent crystal structure of the adenosine A2A receptor with a sodium ion bound. Chapters 3 and 4 complement the crystal structure with additional results from combined biochemistry, biophysical, molecular dynamics, and mutational studies. Chapter 5 describes the synthesis of novel amiloride derivatives that bind in the sodium ion site but also protrude into the orthosteric binding site. In Chapters 3 to 5, radio-labeled ligands were used to quantify ligand binding to the receptor, and Chapter 6 describes an alternative approach towards ligand binding assays. Instead of using a radio-label, mass spectrometry was used to quantify binding of an unlabeled ligand to adenosine A1 and A2A receptors. Show less
Schistosomiasis is a tropical disease affecting over 230 million people worldwide. Although effective drug treatment is available, reinfections are common, and development of immunity is slow. Most... Show moreSchistosomiasis is a tropical disease affecting over 230 million people worldwide. Although effective drug treatment is available, reinfections are common, and development of immunity is slow. Most antibodies raised during schistosome infection are directed against glycans, some of which are thought to be protective. Developing schistosomula are considered most vulnerable to immune attack, and better understanding of local antibody responses raised against glycans expressed by this life stage might reveal possible glycan vaccine candidates for future vaccine research. In this tehsis we adressed the spatial and temporal expression of glycans expressed during the critical larval stages of schistosome development and we studied the (protective) antibody responses against these glycans in animals and infected human populations. Together these studies thereby contribute to an important basis for the understanding of the anti-glycan antibody responses towards Schistosoma in general and towards the vulnerable schistosomulum in particular. Show less
In this thesis mass spectrometry based protein profiling was applied as a new biomarker screening modality and it was evaluated whether or not this could be translated into early detection... Show more In this thesis mass spectrometry based protein profiling was applied as a new biomarker screening modality and it was evaluated whether or not this could be translated into early detection of breast cancer and pancreatic cancer. The status of breast cancer screening by proteomic profiling is discussed. Which steps have already been made? What is essential to implement this techniques in a clinical setting? Furthermore, the new protein profiling screening methods for pancreatic cancers are evaluated. Future studies will be suggested that are needed to translate this promising biomarker into a clinical application. Show less
Recent developments in the coupling of liquid phase separations and mass spectrometry have yielded some new source designs for electrospray ionization. The research in this thesis covers the... Show moreRecent developments in the coupling of liquid phase separations and mass spectrometry have yielded some new source designs for electrospray ionization. The research in this thesis covers the application of a newly developed electrospray ionization source design and it's application for the coupling of capillary electrophoresis to mass spectrometry. The initial chapters cover improvements in sensitivity and separation power in capillary electrophoresis mass spectrometry (CE-MS) by allowing electrospray at ultra low flow rates and therefore optimal ionization and separation conditions. We showed strong complementarity of this CE-MS system with reversed phase liquid chromatography in the identification of peptides and proteins from e.coli digests. Finally a sample preparation method for peptide and protein identification from laser micro-dissected tissue samples from human kidneys was developed. It was shown that a few hundred proteins could be identified from amounts of material representative of one full human glomerulus. On the whole great strides were made in development of strategies employing CE-MS for proteomics analysis. Show less
The involvement of Minor histocompatibility antigens (MiHA) in the graft versus tumor is described about three decades ago. Nowadays, there are many evidences that, after HLA-matched allogeneic... Show moreThe involvement of Minor histocompatibility antigens (MiHA) in the graft versus tumor is described about three decades ago. Nowadays, there are many evidences that, after HLA-matched allogeneic hematopoietic stem cell transplantation (HSCT) MiHA specific T cells mediate both graft versus leukemia (GVL) reactivity and graft versus host disease (GVHD), which is a major cause of morbidity and mortality. To identify MiHA we decided to establish a new strategy based on the bone fide eluted ligandome. Then we developed a database dedicated for identification of polymorphic peptides called human short peptide variation database (HSPVdb). To identify potential MiHA among the huge number of polymorphic peptides eluted from HLA-A*0201 or HLA-B*0702 we selected the top 25 MiHA candidates identified in chapter 2. From this set of data we could validate 2 novel potential MiHA being (LB-CLYBL-1Y & LB-TEP1-1S). To find out at which stages the great_est losses occurred, we developed a method to be able to quantitate MiHA on the cell surface. In addition we studied the phenomenon of the unidirectional T cell responses against MiHA. We studied in depth the presentation of non-canonical long peptides in HLA-A*0201 molecules. Show less
T-cell recognition of MiHA plays an important role in the GVT effect of allo-SCT. Selective infusion of T-cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA-class I... Show moreT-cell recognition of MiHA plays an important role in the GVT effect of allo-SCT. Selective infusion of T-cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA-class I molecules may help to separate the beneficial GVT effects from GVHD after allo-SCT. To date, only a few MiHA that form attractive targets for adoptive immunotherapy have been characterized and the number of patients that can be treated with such MiHA-selective cell therapy remains limited. In this thesis we focused on __reverse immunology__ as an attractive strategy to identify clinically relevant MiHA and other T-cell epitopes. In this approach peptide predictions are the starting point and peptide candidates are subsequently screened for their capacity to induce a T-cell response. We investigated the feasibility of computational genome-wide prediction of hematopoietic MiHA and alternatively implemented mass spectrometry based HLA-peptidomics as source for candidate peptides. T-cells that reacted with these antigens were collectively isolated by MHC-tetramer pull down. Subsequently, the composition of MHC-tetramer positive T-cell populations was characterized and tested for reactivity against any of the predicted epitopes that was included in the initial MHC-tetramer panel. We generated an algorithm that could be exploited to selectively target T-cells specific for clinically relevant MiHA. Show less
The aim of the research described in this thesis is to study the chemical mechanisms responsible for protein aggregation induced by metal catalyzed oxidation and to investigate the relationship... Show moreThe aim of the research described in this thesis is to study the chemical mechanisms responsible for protein aggregation induced by metal catalyzed oxidation and to investigate the relationship between protein oxidation, aggregation and immunogenicity. To this end, recombinant human insulin rhIFN_-1a and rhIFN_-2a are used in conjunction with transgenic mice immune tolerant for the respective human endogenous counterparts. The impact of PEGylation on aggregate formation and excipients to prevent metal catalyzed oxidation are also evaluated. Show less
Schistosomes are parasitic helminths that cause chronic infections in over 200 million people in tropical and sub-tropical areas around the world. Glycoproteins from the eggs of the parasite... Show moreSchistosomes are parasitic helminths that cause chronic infections in over 200 million people in tropical and sub-tropical areas around the world. Glycoproteins from the eggs of the parasite Schistosoma mansoni induce various immune responses in the human host, including T-cell modulation and granuloma formation. Three major, immunogenic egg glycoproteins have been identified; omega-1, IPSE/_1 and kappa-5. The studies in this thesis have unraveled structural and molecular details of the interaction between these three glycoproteins and innate immune cells of the host. Firstly, we have studied the structural details of the N-glycans expressed on these three glycoproteins. Most notably, omega-1 and IPSE/_1 carry diantennary N-glycans which mainly display Lewis X motifs, while kappa-5 expresses triantennary N-glycans with LDN motifs. Then, we have investigated the interaction of the egg glycoproteins with C-type lectin receptors on antigen-presenting immune cells. We show that specific glycan motifs on the egg glycoproteins determine differential binding to these lectin receptors. Finally, we have investigated functional effects of omega-1 and kappa-5. We show that the Th2-modulating capacity of omega-1 is dependent on both glycan-lectin interactions as well as its RNase activity. We found that kappa-5 contains granuloma-inducing properties, which are partly mediated by its LDN motifs. Show less
