Therapeutic proteins have been successfully developed for advancing medical treatments. They are usually large molecules produced by host cells and have a high degree of complexity compared to... Show moreTherapeutic proteins have been successfully developed for advancing medical treatments. They are usually large molecules produced by host cells and have a high degree of complexity compared to synthetic small molecule-based therapeutics. The complexity is mainly attributed to the heterogenic nature of post-translational modifications (PTMs). Glycosylation is one of the main drivers of protein heterogeneity. Since each modification may potentially impact the safety and efficacy, analyticalmethods for the structural and functional characterization of protein-based therapeutics are highly demanded. This thesis presents novel mass spectrometry (MS)-based methods for the analysis of therapeutic glycoproteins. It covers aspects of glycobioinformatics and sample preparation for improved bottom-up glycoproteomic analysis. Further, the profiling of complex intact glycoproteins by matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS is demonstrated. Finally, Fc gamma receptor III affinity chromatography combined with online MS detection is presented for novel proteoform-resolved structure-function insights of monoclonal antibodies. Show less
The interaction between tumor and immune cells in the microenvironment plays a key role in oncogenesis. This recognition can be mediated by changes in glycosylation on tumor cells which are sensed... Show moreThe interaction between tumor and immune cells in the microenvironment plays a key role in oncogenesis. This recognition can be mediated by changes in glycosylation on tumor cells which are sensed by lectins, such as the Macrophage Galactose-type Lectin (MGL), expressed on immune cells, resulting in immunosuppressive responses. MGL binding to the Tn-epitope on MUC1 triggers DCs to stimulate T-regulatory responses and tolerance against the tumor, while suppressing T-effector cells responsible for tumor eradication. This undesirable consequence in cancer led to interest in the characterization of targets of MGL. This thesis dealt with the analysis of glycoproteins binding to MGL expressed by different tumor cell models. For this purpose, a robust method to enrich MGL binding proteins, in combination with their mass spectrometry-based identification was established. Because of the high level of the Tn-antigen, due to a Cosmc mutation, the Jurkat cell line was initially used for this purpose (Chapter 2) but a slightly adjusted method was later applied to high- and low-MGL binding CRC cell lines as well (Chapter 3 and 5). These analyses led to the identification of hitherto unknown MGL binders. In Chapter 4, we focused on to the contribution of N-glycan MGL binding glycotopes in CRC cell lines, most probably corresponding to LacdiNAc structures, by implementing overall N-glycan release in our workflows. With this, we were able to show that N-glycoproteins represent a hitherto underestimated group of MGL binding protein in these cell lines. Also several secreted proteins from the CRC cell lines could bind to MGL (Chapter 5), indicating that the interaction with immune cells can also be mediated by this group of proteins. The results from previously published transcriptomics and N-/O-glycomic analyses could not explain the different expression of MGL binding proteins on the CRC cell lines used. For this reason, in Chapter 4, we extended our research with full comparative quantitative proteomics analyses, in an attempt to explain the differences in MGL binding, for example by different levels of MGL binding proteins or proteins involved in glycosylation pathways. Additionally, in Chapter 6, we used such a quantitative proteomics dataset also to test the suitability of a previously suggested mass spectrometry-based method to discriminate O-GalNAc (Tn) versus O-GlcNAc, which led to the first site-specific identification of O-glycosylation of both intracellular and secreted anterior gradient protein 2 (AGR2). Show less
Colorectal cancer (CRC) is the second-leading cause of cancer death worldwide due in part to a high proportion of patients diagnosed at advanced stages of the disease. For this reason, many efforts... Show moreColorectal cancer (CRC) is the second-leading cause of cancer death worldwide due in part to a high proportion of patients diagnosed at advanced stages of the disease. For this reason, many efforts have been made towards new approaches for early detection and prognosis. Cancer-associated aberrant glycosylation, especially the Tn and STn antigens, can be detected using the macrophage galactose-type C-type lectin (MGL/CLEC10A/CD301), which has been shown to be a promising tool for CRC prognosis. We had recently identified the major MGL-binding glycoproteins in two high-MGL-binding CRC cells lines, HCT116 and HT29. However, we failed to detect the presence ofO-linked Tn and STn glycans on most CRC glycoproteins recognized by MGL. We therefore investigated here the impact ofN-linked andO-linked glycans carried by these proteins for the binding to MGL. In addition, we performed quantitative proteomics to study the major differences in proteins involved in glycosylation in these cells. Our results showed thatN-glycans have a significant, previously underestimated, importance in MGL binding to CRC cell lines. Finally, we highlighted both common and cell-specific processes associated with a high-MGL-binding phenotype, such as differential levels of enzymes involved in protein glycosylation, and a transcriptional factor (CDX-2) involved in their regulation. Show less
Background: The Ca2+-dependent C-type lectin receptor Macrophage Galactose-type Lectin (MGL) is highly expressed by tolerogenic dendritic cells (DC) and macrophages. MGL exhibits a high binding... Show moreBackground: The Ca2+-dependent C-type lectin receptor Macrophage Galactose-type Lectin (MGL) is highly expressed by tolerogenic dendritic cells (DC) and macrophages. MGL exhibits a high binding specificity for terminal alpha- and beta-linked GaINAc residues found in Tn, sTn and LacdiNAc antigens. These glycan epitopes are often overexpressed in colorectal cancer (CRC), and, as such, MGL can be used to discriminate tumor from the corresponding healthy tissues. Moreover, the high expression of MGL ligands is associated with poor diseasefree survival in stage III of CRC tumors. Nonetheless, the glycoproteins expressed by tumor cells that are recognized by MGL have hitherto remained elusive.Methods: Using a panel of three CRC cell lines (HCT116, HT29 and LS174T), recapitulating CRC diversity, we performed FACS staining and pull-down assays using a recombinant soluble form of MGL (and a mutant MGL as control) combined with mass spectrometry-based (glyco)proteomics.Results: HCT116 and HT29, but not LS174T, are high MGL-binding CRC cell lines. On these cells, the major cell surface binding proteins are receptors (e.g. MET, PTK7, SORL1, PTPRF) and integrins (ITGB1, ITGA3). From these proteins, several N- and/or O-glycopeptides were identified, of which some carried either a LacdiNAc or Tn epitope.Conclusions: We have identified cell surface MGL-ligands on CRC cell lines.General significance: Advances in (glyco)proteomics have led to identification of candidate key mediators of immune-evasion and tumor growth in CRC. Show less
The aim of this thesis is to explore the glycosylation of PSA as well as to study if alterations can be observed between patients with indolent and malignant PCa. For this purpose the powerful... Show moreThe aim of this thesis is to explore the glycosylation of PSA as well as to study if alterations can be observed between patients with indolent and malignant PCa. For this purpose the powerful analytical platform CE-ESI-MS(/MS) was explored with a special focus on the analysis of glycans and glycopeptides (Chapter 1). The first section of the thesis describes the different method developments implemented for the analysis of PSA with CE-ESI-MS. Namely, Chapter 2 describes that CE-ESI-MS enables to separate glycopeptides with differently linked sialic acids without any additional sample treatment, Chapter 3 shows that an introduction of a dopant enriched nitrogen gas improves the limit of detection (sensitivity) of glycopeptides and Chapter 4 introduces a novel labeling procedure of total plasma N-glycome with the hydrazide Girard’s reagent P. Chapter 5 describes the development of a PSA Glycomics Assay which allows the capture of intact PSA from patients’ urine followed by analysis with the optimized CE-ESI-MS platform (Chapters 2 and 3). Finally, Chapter 6 offers a general discussion about future developments, the potential of PSA glycosylation in the clinical setting, showing the relevance of our results and how these may contribute to further clinical applications towards personalized medicine. 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
Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be... Show moreGlycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level. Show less
In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses... Show moreIn-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying > 110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control. Show less
The application of dedicated mass spectrometry (MS) and nuclear magnetic resonance (NMR) technologies for biomarker discovery and diagnostic purposes has increased substantially in the last decade.... Show moreThe application of dedicated mass spectrometry (MS) and nuclear magnetic resonance (NMR) technologies for biomarker discovery and diagnostic purposes has increased substantially in the last decade. In the studies presented in this thesis, we have used these technologies to identify parasite or host-derived products (biomarkers) related to infection and morbidity associated with schistosomiasis and to better understand the host-parasite interaction. The application of our peptidomics and metabonomics studies on schistosomiasis have provided some novel, valuable information but they are obviously only the first step. In addition to the potential biomarkers identified with the global biomarker discovery approaches, we showed in this thesis that a more targeted approach, looking at glycosylation, also resulted in novel information on S. mansoni infection. We have identified and characterized a set of human Apolipoprotein C-III peptides aberrantly glycosylated at the O-glycosylation site (Thr74), in urine of S. mansoni- infected individuals. The presented study is the first in which MS and NMR were used for the analysis of a cohort of human S. mansoni-infected individuals. This has resulted in the identification of a number of novel markers. Nevertheless, further studies are needed to evaluate the overall applicability of these putative biomarkers Show less
