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
In this thesis we explore the Ubiquitin code using purification methods coupled with mass spectrometry. We overview the available methods and current knowledge of of Ubiquitin and SUMO... Show moreIn this thesis we explore the Ubiquitin code using purification methods coupled with mass spectrometry. We overview the available methods and current knowledge of of Ubiquitin and SUMO modifications with examples of substrates where the precise site of modification is important, and substrates where the modification site seems interchangeable.We show that the deubiquitinating enzymes regulate a separate subset of the ubiquitinome than the proteasome. The PARylating enzyme PARP1 show increased activity after ubiquitination, which is regulated by deubiquitinating enzymes.Furthermore, we developed an in-vivo tool to study SUMO dependent interaction using proximity labeling which can be used for microscopy or identification by mass spectrometry.Finally, we investigate binders of Ubiquitin and SUMO chains with specific linkages and elucidate a preference for some chain linkages based on biological pathway of the binder. We also explore possible binding domains and predict the structure of some chain binding complexes using protein docking. Show less
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
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
Riedo, A.; Grimaudo, V.; Aerts, J.W.; Lukmanov, R.; Tulej, M.; Broekmann, P.; ... ; Ehrenfreund, P. 2021
The work presented in this thesis describes the improvement and application of on-tissue chemistry for in-situ biomolecular analysis using matrix assisted-laser desorption/ionization mass... Show moreThe work presented in this thesis describes the improvement and application of on-tissue chemistry for in-situ biomolecular analysis using matrix assisted-laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We have proposed new methodologies, applying on-tissue (enzymatic) chemistry, to increase the molecular information obtained in a MALDI-MSI analysis. We have also developed an automated histology-guided MSI platform, based on state-of-the-art image processing tools, to facilitate high mass and spatial resolution MALDI-MSI while maintaining reasonable data loads and acquisition times. We have shown the importance of these methods in a clinical biomarker discovery study on myxoid liposarcoma tissues. Show less
Mirzaian, M.; Wisse, P.; Guimaraes Da Lomba Ferraz, M.J.; Marques, A.R.A.; Gaspar, P.; Oussoren, S.V.; ... ; Aerts, J.M.F.G. 2017
The development of a capillary ion chromatograph is described together with a matching desalter. This desalter made it possible to use on-line a mass spectrometer. The mass spectrometer enables... Show moreThe development of a capillary ion chromatograph is described together with a matching desalter. This desalter made it possible to use on-line a mass spectrometer. The mass spectrometer enables partly to characterize carbohydrates eluting from the anion exchange column. This separation technology is named cap-HPAEC-PAD-MS. The developed capillary ion chromatograph is successfully used to characterize glycan structures in human body fluids, such as urine and amniotic fluid, from individuals suffering form a lysosomal disorder Show less
The aim of the thesis was to develop metabolic analytical platforms for static and dynamic measurements that could answer biological questions for in vitro and in vivo animal models in the area of... Show moreThe aim of the thesis was to develop metabolic analytical platforms for static and dynamic measurements that could answer biological questions for in vitro and in vivo animal models in the area of lipid research. Gene profiling together with the transcriptome and metabolome data was used in combination with the LC/MS analytical platform. In terms of the analytical platforms developed, the focus was on high resolution LC/MS but not limited, as amalgamation with other platforms such as gradient gel electrophoresis (GGE) and fast protein liquid chromatography (FPLC) were explored in more detail to investigate the lipid composition of lipoprotein particles. These analytical strategies were applied to different lipid modulating biological targets as a mean to obtaining a more detailed and characteristic phenotype description directing decisions in drug search during the drug discovery process on the basis of the analytical results obtained. Additionally, the utilization of metabolic tracers was investigated further to probe dynamic changes in the biological target and animal models in question Show less
