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
Kemna, M.J.; Plomp, R.; Paassen, P. van; Koeleman, C.A.M.; Jansen, B.C.; Damoiseaux, J.G.M.C.; ... ; Wuhrer, M. 2017
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
Wesdorp, D.J.W.; Knoester, M.; Braat, A.E.; Coenraad, M.J.; Vossen, A.C.T.M.; Claas, E.C.J.; Hoek, B. 2013
Myasthenia gravis (MG) is hallmarked by acquired and fluctuating weakness of voluntary muscles. In the majority of patients, weakness is caused by autoantibodies to the postsynaptic acetylcholine... Show moreMyasthenia gravis (MG) is hallmarked by acquired and fluctuating weakness of voluntary muscles. In the majority of patients, weakness is caused by autoantibodies to the postsynaptic acetylcholine receptor (AChR) in the neuromuscular junction. Approximately 10% of MG patients are seronegative (SNMG). In 2001, antibodies to muscle-specific kinase (MuSK) were discovered within this group. This dissertation describes the epidemiology, clinical characteristics and immunological aspects of this new disease. In the Netherlands, MuSK MG is rare. Incidence was 0.17 per million person-years. Prevalence was 2.8 per million on January 1st 2004. Weakness was more often bulbar, and lead to frequent respiratory crises. MuSK MG was linked to the HLA-DR14-DQ5 haplotype and the disease severity was associated to antigen-specific IgG4 antibodies instead of IgG1. This is in contrast to AChR MG in which autoantibodies are mainly of the IgG1 and IgG3 subclass, and the disease is linked to HLA-B8-DR3. In SNMG patients, no antibodies to postsynaptic ErbB receptors were found. A case-report describes the transmission of MuSK autoantibodies from mother to her newborn child, causing transient weakness in the infant. A second case-report illustrates the effect of MuSK antibodies on both pre- and postsynaptic signal transmission in the neuromuscular junction. Show less
