MSACL 2019 EU Abstract

IgG and IgA Glycopeptide Characterization by LC-MS Reveals Associations with Inflammatory Bowel Disease Subtypes and Behavior

Noortje de Haan (1), Rosina Plomp (1), Ana Momcilovic (1), Mirna Simurina (2), Frano Vuckovic (2), Florent Clerc (1), Gordan Lauc (2), Viktoria Dotz (1), Manfred Wuhrer (1)
(1) Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands, (2) Genos Glycoscience Research Laboratory, BIOCentar, Zagreb, Croatia

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Noortje de Haan (Presenter) Leiden University Medical Center

Presenter Bio: My name is Noortje de Haan, I am conducting my post-doctoral research at the Center for Proteomics and Metabolomics at the Leiden University Medical Center, The Netherlands. In 2014, I obtained my MSc degree in Pharmaceutical Sciences at the VU University Amsterdam, The Netherlands, with a focus on biomarker research and clinical chemical analysis. Last February, I received my PhD at the Leiden University Medical Center, under the supervision of prof. Manfred Wuhrer, on the development and application of various mass spectrometry-based methods for the analysis of (antibody) glycosylation. My enthusiasm for glycoproteomic-related research started early in my scientific career and this remains a key drive in my current work. Furthermore, I am interested in the development of mass spectrometric methods and data analysis protocols for addressing clinical research questions.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Introduction
Inflammatory bowel diseases (IBDs) are prevalent diseases with a high impact on the quality of life of the patients. Causes of IBD are not well understood and the most prominent forms, Crohn’s disease (CD) and ulcerative colitis (UC), are sometimes hard to distinguish. Immunoglobulin (Ig) G and IgA play a prominent role in IBD pathogenesis and their effector functions are known to be influenced by the glycosylation of the constant domain. Here, we developed methods for the site-specific profiling of IgG and IgA glycosylation in plasma and saliva and applied them to study bio fluid-specific antibody glycosylation profiles in healthy volunteers as well as IBD associated changes in IBD patients.

Methods
IgG and IgA were isolated from human plasma and saliva by bead-based affinity chromatography. The purified proteins were digested by trypsin and immunoglobulin subclass-specific glycoprofiling was performed based on the mass spectrometric detection of glycopeptides. IgG and IgA were characterized in 3441 plasma samples obtained from two independent cohorts of patients with CD or UC and healthy controls.
For the saliva-derived IgG and IgA, samples were analyzed from 19 healthy volunteers and comparisons were made between glycosylation profiles from plasma- and saliva-derived antibodies from the same individual.


Results
In patients suffering from IBD, we found lower IgG galactosylation, a well-known marker for systemic inflammation, for different types of IBD as compared to healthy controls. Furthermore, this glycosylation trait was associated with disease severity. Interestingly, the level of various glycosylation features on both IgG and IgA was different between CD and UC. Additionally, we found IgG glycosylation to associate with disease behavior and progression.
The method developed for the analysis of salivary antibodies enabled us to assess the N- and O-glycosylation of all glycosylation sites of both plasma- and saliva-derived IgA, including the one on the joining chain of dimeric IgA. Additionally, for salivary IgA the glycosylation of the associated secretory component was characterized. Salivary IgA exhibited a substantially different glycosylation, as compared to plasma IgA in healthy controls.

Discussion
Based on the differences in antibody glycosylation found between different types of IBD and the associations with disease characteristics, we expect a role for Ig glycosylation markers in IBD classification, the prediction of disease prognosis, and for treatment monitoring.
The characterization of salivary IgA was, as of yet, not applied in disease cohorts. As IgA is a well-known mucosal antibody and we here show bio fluid-specific glycosylation profiles for this protein, we expect the developed method for salivary IgA glycosylation profiling to be particularly relevant for diseases with mucosal involvement, such as IBD and colon cancer.