Our glyco(proteo)mics research is performed in close collaboration with several clinical departments, both within the LUMC and elsewhere. With the department of Rheumatology at the LUMC we are working on basic (immuno)glycobiology, as well as the sensitive glycosylation analysis of autoantibodies, in particular ACPAs, showing more pronounced inflammatory Fc glycosylation profiles, and extraordinary high levels of Fab glycosylation.1-3
Another fruitful collaboration has been with the department of Rheumatology at the Erasmus MC. Within this collaboration we have confirmed a clear association of IgG glycosylation changes with the improvement of rheumatoid arthritis disease activity during pregnancy.4
Recent technical advances have allowed for improved detection of sialic acids by LC-MS5 and MALDI-MS6,7. Using these methods we have been able to address delicate and subtle biological questions, for example regarding the potential roles of galactose and sialic acid in inflammation, using arthritis disease activity as a readout.
Inflammatory bowel disease
The inflammatory bowel diseases (IBD) Crohn’s disease (CD) and ulcerative colitis (UC) are conditions affecting the intestinal tract, with a high impact on patients daily life. They are considered autoimmune diseases, however the precise etiology and origins still remain unknown. The search for specific and sensitive biomarkers to help distinguishing CD from UC in an early stage would be an immense benefit for the treatment of IBD patients.
As part of the European Inflammatory Bowel Disease Biomarkers Programme (IBD BIOM) and in collaboration with the university hospital of Careggi, the Cedars-Sinai hospital of Los Angeles and Genos Ltd in Zagreb, we are searching for glycomic IBD biomarkers in the total plasma N-glycome and specifically on the protein Immunoglobulin G (IgG) in samples of more than 3000 patients and healthy controls.
Practically, we are making use of our automated preparation platform to release the glycan moiety from proteins and we modify their sialic acids to obtain sialic acid linkage-specific information.8 The samples are then analysed with our previously established matrix-assisted laser desorption-ionization time-of-flight mass spectrometric platform (MALDI-TOF-MS).6 In addition, we isolate IgG from patient plasma and analyse protein specific glycopeptides on a liquid chromatography (LC)-TOF-MS platform.
Allergy and immunoglobulin E
Of all the immunoglobulins, immunoglobulin E (IgE) has the capacity to trigger the strongest immunologic reactions. While it provides protection against parasitic infections, IgE is mainly known for its role as the mediator of allergic responses. IgE is also the most glycosylated antibody (roughly 12% of the total mass), but due to its low abundance in serum (<1 µg/mL) research into IgE glycosylation has been sparse.9
At the Center for Proteomics and Metabolomics, we have developed a site-specific method to analyze the six N-glycosylation sites of IgE using various proteolytic enzymes and liquid chromatography-mass spectrometry.10 Using this method, we found that the IgE N-glycosylation of a hyperimmune donor was similar to that of healthy individuals, while the N-glycosylation of IgE from a myeloma patient showed clear differences.
- Rombouts, Y. et al. Extensive glycosylation of ACPA-IgG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis. Ann. Rheum. Dis. 75, 578-585 (2016).
- Rombouts, Y. et al. Anti-citrullinated protein antibodies acquire a pro-inflammatory Fc glycosylation phenotype prior to the onset of rheumatoid arthritis. Ann. Rheum. Dis. 74, 234-241 (2015).
- Wang, J. et al. Fc-glycosylation of IgG1 is modulated by B-cell stimuli. Mol. Cell. Proteomics 10, M110 004655 (2011).
- Bondt, A. et al. Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation. J. Proteome Res. 12, 4522-4531 (2013).
- Selman, M. H. J. et al. Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface. J. Proteomics 75, 1318-1329 (2012).
- Reiding, K. R., Blank, D., Kuijper, D. M., Deelder, A. M. & Wuhrer, M. High-Throughput Profiling of Protein N-Glycosylation by MALDI-TOF-MS Employing Linkage-Specific Sialic Acid Esterification. Anal. Chem. 86, 5784-5793 (2014).
- de Haan, N. et al. Linkage-specific sialic acid derivatization for MALDI-TOF-MS profiling of IgG glycopeptides. Anal. Chem. 87, 8284-8291 (2015).
- Bladergroen, M. R. et al. Automation of high-throughput mass spectrometry-based plasma N-glycome analysis with linkage-specific sialic acid esterification. J. Proteome Res. 14, 4080-4086 (2015).
- Plomp, R., Bondt, A., de Haan, N., Rombouts, Y. & Wuhrer, M. Recent Advances in Clinical Glycoproteomics of Immunoglobulins (Igs). Mol. Cell. Proteomics 15, 2217-2228 (2016).
- Plomp, R. et al. Site-specific N-glycosylation analysis of human immunoglobulin e. J. Proteome Res. 13, 536-546 (2014).