Protein glycosylation has profound implications in a wide range of molecular and biological processes occurring in cancer, where specific changes in the glycan structures have shown to be... Show moreProtein glycosylation has profound implications in a wide range of molecular and biological processes occurring in cancer, where specific changes in the glycan structures have shown to be associated with the development and progression of the disease paving the way for the development of new clinical biomarkers as well as providing specific targets for therapeutic intervention, patient stratification and personalized medicine. Protein glycosylation is also critical for the development of biopharmaceuticals, as even minor shifts in manufacturing procedures can substantially impact the bioactivity, safety, and efficacy of therapeutic proteins. Although a variety of mass spectrometric and chromatographic methods are available for the identification and characterization of glycans from complex sample mixtures, the lack of standardized protocols across platforms often results in inconsistent results, making data integration and comparison challenging. Furthermore, most of the current technology for the study of intact glycans would not be suitable for the rapid analysis of large sample sets, mainly due to limitations in sample throughput. The scope of this thesis is to establish standardized, high-throughput glycomics technologies for the quantitative analysis of protein N- and O-glycosylation and improve current methodologies in order to facilitate the characterization of intact oligosaccharides from in vitro established model systems. 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
Antibody-based drugs are the largest class of biopharmaceuticals produced. They are currently employed to treat diverse diseases such as cancer, cardiovascular or neuronalogical diseases. Whereas... Show moreAntibody-based drugs are the largest class of biopharmaceuticals produced. They are currently employed to treat diverse diseases such as cancer, cardiovascular or neuronalogical diseases. Whereas some years ago, efforts were focused on developing classical monoclonal antibodies (mAbs), nowadays there is a trend towards engineered antibodies with enhanced functions, such as bispecific antibodies (BsAbs). These molecules increase on structural complexity and bring new challenges for their analytical characterization. Furthermore, the continuous development of new antibody treatments and molecules also increases the pressure in the analytical labs longing for more automation and high-throughput mass spectrometric analysis. This thesis addresses these aspects and provide solutions for improved and efficient characterization of novel antibody therapeutics focusing on mass spectrometric approaches. These approaches are based on multidimensional liquid chromatography or capillary electrophoresis hyphenated to mass spectrometry or on the application of in-source decay fragmentation. Next to structural characterization, in this thesis MS-based approaches have been developed for their functional characterization permitting to study the relationships between antibody structure and function. 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
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
The cannabis plant (Cannabis sativa L.) has a long history as a recreational drug, but also as part of traditional medicine in many cultures. Nowadays, it is used by a large number of patients... Show moreThe cannabis plant (Cannabis sativa L.) has a long history as a recreational drug, but also as part of traditional medicine in many cultures. Nowadays, it is used by a large number of patients worldwide, to ameliorate the symptoms of diseases varying from cancer and AIDS to multiple sclerosis and migraine. The discovery of cannabinoid-receptors and the endocannabinoid system have opened up a new and exciting field of research. But despite the pharmaceutical potential of cannabis, its classification as a narcotic drug has prevented the successful development of cannabis into modern medicine. Although a huge number of scientific papers has been published on cannabis, there is currently no scientific consensus on the usefulness of medicinal cannabis. In 2004, The Netherlands became the first country to make herbal cannabis available as a prescription drug. The phytochemical research presented in this thesis has been possible because of the availability of these high-grade cannabis plants. This thesis has a specific focus on the cannabinoids and on analytical problems that currently obstruct advanced study of the cannabis plant. Furthermore, it deals with much needed methods for quality control and with administration forms of medicinal cannabis. In general, it may be considered a general guidebook, covering all the basic phytochemical aspects of medicinal cannabis. Show less
