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
Proteins are widely known as key players that fulfill crucial roles at the molecular level in the human body but also for their involvement in many processes in everyday life. For example, proteins... Show moreProteins are widely known as key players that fulfill crucial roles at the molecular level in the human body but also for their involvement in many processes in everyday life. For example, proteins can be used as medicine in health care or for their enzymatic function in the food industry. All these proteins do not exist as a single species but rather as a complex mixture of structural variants, so-called proteoforms. This heterogeneity results mainly from the presence of post-translational modifications (PTMs), such as glycosylation and glycation. To further complicate this matter, these PTMs can induce structural as well as functional changes. To allow in-depth structural and functional characterization of these proteoforms, novel analytical approaches are required to resolve proteoform heterogeneity while persevering protein nativity. The hyphenation of native separation techniques with mass spectrometry has emerged as a powerful approach to reliably study these aspects. The work in this thesis describes the (further) development and application of such methodologies for biopharmaceutical and biotechnological products. Show less