Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well... Show moreChanges in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson's r correlation coefficient of 0.8208 between the two methods. 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
Simurina, M.; Haan, N. de; Vuckovic, F.; Kennedy, N.A.; Stambuk, J.; Falck, D.; ... ; Inflammatory Bowel Dis Biomarkers 2018
