Albert Bondt

Glycoproteomic Analysis of Antibodies

Antibody glycosylation has been shown to change with various processes. This review presents mass spectrometric approaches for antibody glycosylation analysis at the level of released glycans, glycopeptides, and intact protein. With regard to IgG fragment crystallizable glycosylation, mass spectrometry has shown its potential for subclass-specific, high-throughput analysis. In contrast, because of the vast heterogeneity of peptide moieties, fragment antigen binding glycosylation analysis of polyclonal IgG relies entirely on glycan release. Next to IgG, IgA has gained some attention, and studies of its O- and N-glycosylation have revealed disease-associated glycosylation changes. Glycoproteomic analyses of IgM and IgE are lagging behind but should complete our picture of glycosylations influence on antibody function.

Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation

Rheumatoid arthritis (RA) is known to improve during pregnancy and to flare after delivery. Changes in the glycosylation of immunoglobulin G (IgG)s fragment crystallizable (Fc) have been suggested to play a role herein. Recent animal studies indicate that not galactosylation but mainly sialylation is important in this respect. We aim to find new associations between IgG-Fc N-glycosylation and improvement of RA during pregnancy and the flare after delivery. Sera of RA patients (n = 251 pregnancies) and healthy controls (n = 32), all participating in a prospective cohort study on RA and pregnancy (PARA study), were collected before conception, during pregnancy, and after delivery. Using a recently developed fast and robust nanoRP-HPLC-sheath-flow-ESI-MS method the glycosylation of IgG Fc-glycopeptides was measured in a subclass specific manner, with relative standard deviations of <4% for the 8 most abundant IgG Fc glycopeptides during the entire measurement period of over 3 weeks. In patients and controls, several glycosylation changes were observed during pregnancy. In depth analysis of the association of these glycosylation changes with disease activity revealed that galactosylation, independent of sialylation, is associated with improvement of RA during pregnancy. Functional studies in human cell systems should be performed to obtain more insight into this matter.

Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface

Biological activities of immunoglobulin G such as effector functions via Fc receptor interactions are influenced by Fc-linked N-glycans. Here we describe a fast, robust and sensitive nano-LC-ESI-MS method for detailed subclass specific analysis of IgG Fc N-glycosylation. A sheath-flow ESI sprayer was used as a sensitive zero dead volume plug-and-play interface for online MS coupling, generating a very constant spray and ionization over the entire LC gradient. The propionic acid containing sheath-liquid effectively suppressed TFA gas-phase ion-pairing, enabling the use of TFA containing mobile phases. The fixed position of the sheath-flow ESI sprayer, far away from the glass capillary inlet, reduced MS contamination as compared to conventional nano-ESI. The method was found to be suitable for fast and detailed subclass specific IgG Fc N-glycosylation profiling in human plasma. The obtained subclass specific IgG Fc N-glycosylation profiles were processed automatically using in house developed software tools. For each of the IgG subclasses the 8 major glycoforms showed an interday analytical variation below 5%. The method was used to profile the IgG Fc N-glycosylation of 26 women at several time points during pregnancy and after delivery, revealing pregnancy-associated changes in IgG galactosylation, sialylation and incidence of bisecting N-acetylglucosamine.

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding.

Human plasma protein N-glycosylation.

Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level.

Automation of High-Throughput Mass Spectrometry-Based Plasma N-Glycome Analysis with Linkage-Specific Sialic Acid Esterification

Glycosylation is a post-translational modification of key importance with heterogeneous structural characteristics. Previously, we have developed a robust, high-throughput MALDI-TOF-MS method for the comprehensive profiling of human plasma N-glycans. In this approach, sialic acid residues are derivatized with linkage-specificity, namely the ethylation of α2,6-linked sialic acid residues with parallel lactone formation of α2,3-linked sialic acids. In the current study, this procedure was used as a starting point for the automation of all steps on a liquid-handling robot system. This resulted in a time-efficient and fully standardized procedure with throughput times of 2.5 h for a first set of 96 samples and approximately 1 h extra for each additional sample plate. The mass analysis of the thus-obtained glycans was highly reproducible in terms of relative quantification, with improved interday repeatability as compared to that of manual processing.

Recent Advances in Clinical Glycoproteomics of Immunoglobulins (Igs)

Antibody glycosylation analysis has seen methodological progress resulting in new findings with regard to antibody glycan structure and function in recent years. For example, antigen-specific IgG glycosylation analysis is now applicable for clinical samples because of the increased sensitivity of measurements, and this has led to new insights in the relationship between IgG glycosylation and various diseases. Furthermore, many new methods have been developed for the purification and analysis of IgG Fc glycopeptides, notably multiple reaction monitoring for high-throughput quantitative glycosylation analysis. In addition, new protocols for IgG Fab glycosylation analysis were established revealing autoimmune disease-associated changes. Functional analysis has shown that glycosylation of IgA and IgE is involved in transport across the intestinal epithelium and receptor binding, respectively.

Pregnancy-associated serum N-glycome changes studied by high-throughput MALDI-TOF-MS

Pregnancy requires partial suppression of the immune system to ensure maternal-foetal tolerance. Protein glycosylation, and especially terminal sialic acid linkages, are of prime importance in regulating the pro- and anti-inflammatory immune responses. However, little is known about pregnancy-associated changes of the serum N-glycome and sialic acid linkages. Using a combination of recently developed methods, i.e. derivatisation that allows the distinction between α2,3- and α2,6-linked sialic acids by high-throughput MALDI-TOF-MS and software-assisted data processing, we analysed the serum N-glycome of a cohort of 29 healthy women at 6 time points during and after pregnancy. A total of 77 N-glycans were followed over time, confirming in part previous findings while also revealing novel associations (e.g. an increase of FA2BG1S1(6), FA2G1S1(6) and A2BG2S2(6) with delivery). From the individual glycans we calculated 42 derived traits. With these, an increase during pregnancy and decrease after delivery was observed for both α2,3- and α2,6-linked sialylation. Additionally, a difference in the recovery speed after delivery was observed for α2,3- and α2,6-linked sialylation of triantennary glycans. In conclusion, our new high-throughput workflow allowed the identification of novel plasma glycosylation changes with pregnancy.

Structural analysis of variable domain glycosylation of anti-citrullinated protein antibodies in rheumatoid arthritis reveals the presence of highly sialylated glycans.

Recently, we showed the unexpectedly high abundance of N-linked glycans on the Fab-domain of Anti-Citrullinated Protein Antibodies (ACPA). As N-linked glycans can mediate a variety of biological functions, we now aimed at investigating the structural composition of the Fab-glycans of ACPA-IgG to better understand their mediated biological effects. ACPA-IgG and noncitrulline specific (control) IgG from plasma and/or synovial fluid of nine ACPA positive rheumatoid arthritis patients were affinity purified. The N-linked glycosylation of total, Fc and F(ab′)2 fragments, as well as heavy and light chains of ACPA-IgG and control IgG were analyzed by UHPLC and MALDI-TOF mass spectrometry. The Fc-glycosylation of ACPA-IgG and IgG was analyzed at the glycopeptide level using LC-MS. The structural analyses revealed that ACPA-IgG molecules contain highly sialylated glycans in their Fab-domain. Importantly, Fab-glycans were estimated to be present on over 90% of ACPA-IgG, which is five times higher than in control IgG isolated from the same patients. This feature was more prominent on ACPA isolated from synovial fluid compared with peripheral blood. These observations provide the first evidence pointing to the ability of ACPA-IgG to mediate novel immunological activities, for example through binding specific lectins via hyper-sialylated Fab-glycans.

Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy

BackgroundChanges in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation.MethodsIgGs were captured from RA and control sera obtained before (RA only), during and after pregnancy, followed by Fc and Fab separation, glycan release, and mass spectrometric detection. In parallel, glycans from intact IgG were analysed. The data was used to calculate glycosylation traits, and to estimate the level of Fab glycosylation.ResultsThe overall level of Fab glycosylation was increased in RA patients compared to controls, while no differences in Fab glycosylation patterns were found. For the Fc and intact IgG (Total) previously observed differences in galactosylation and bisection were confirmed. Furthermore, increased galactosylation of Fc and Total were associated with lower disease activity and autoantibody positivity. In addition, the change in Fc galactosylation associated with the change in disease activity during pregnancy and after delivery, while this was not the case for Fab.ConclusionsIn contrast to changes in Fc glycosylation, changes in Fab glycosylation are not associated with improvement of RA during pregnancy and arthritis flare after delivery.