Recent Advances in Clinical Glycoproteomics of Immunoglobulins (Igs).
ABSTRACT: 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.
Project description:BACKGROUND:Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are pathogenic in ANCA-associated vasculitis (AAV). The respective role of IgG Fc and Fab glycosylation in mediating ANCA pathogenicity is incompletely understood. Herein we investigate in detail the changes in Fc and Fab glycosylation in MPO-ANCA and Pr3-ANCA and examine the association of glycosylation aberrancies with disease activity. METHODOLOGY:Total IgG was isolated from serum or plasma of a cohort of 30 patients with AAV (14 MPO-ANCA; 16 PR3-ANCA), and 19 healthy control subjects. Anti-MPO specific IgG was affinity-purified from plasma of an additional cohort of 18 MPO-ANCA patients undergoing plasmapheresis. We used lectin binding assays, liquid chromatography, and mass spectrometry-based methods to analyze Fc and Fab glycosylation, the degree of sialylation of Fc and Fab fragments and to determine the exact localization of N-glycans on Fc and Fab fragments. PRINCIPAL FINDINGS:IgG1 Fc glycosylation of total IgG was significantly reduced in patients with active AAV compared to controls. Clinical remission was associated with complete glycan normalization for PR3-ANCA patients but not for MPO-ANCA patients. Fc-glycosylation of anti-MPO specific IgG was similar to total IgG purified from plasma. A major fraction of anti-MPO specific IgG harbor extensive glycosylation within the variable domain on the Fab portion. CONCLUSIONS/SIGNIFICANCE:Significant differences exist between MPO and PR3-ANCA regarding the changes in amounts and types of glycans on Fc fragment and the association with disease activity. These differences may contribute to significant clinical difference in the disease course observed between the two diseases.
Project description:Previous studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian cancer; however, it was unclear to which proteins these glycans belong. We hypothesize that protein-specific glycosylation profiles of the glycans may be more informative of ovarian cancer and can provide insight into biological mechanisms underlying glycan aberration in serum of diseased individuals. Serum samples from women diagnosed with epithelial ovarian cancer (EOC, n = 84) and matched healthy controls (n = 84) were obtained from the Gynecologic Oncology Group. Immunoglobulin (IgG, IgA, and IgM) concentrations and glycosylation profiles were quantified using multiple reaction monitoring mass spectrometry. Differential and classification analyses were performed to identify aberrant protein-specific glycopeptides using a training set. All findings were validated in an independent test set. Multiple glycopeptides from immunoglubins IgA, IgG, and IgM were found to be differentially expressed in serum of EOC patients compared with controls. The protein-specific glycosylation profiles showed their potential in the diagnosis of EOC. In particular, IgG-specific glycosylation profiles are the most powerful in discriminating between EOC case and controls. Additional studies of protein- and site-specific glycosylation profiles of immunoglobulins and other proteins will allow further elaboration on the characteristics of biological functionality and causality of the differential glycosylation in ovarian cancer and thus ultimately lead to increased sensitivity and specificity of diagnosis.
Project description:BACKGROUND:Mycobacterium tuberculosis remains a global health problem and clinical management is complicated by difficulty in discriminating between latent infection and active disease. While M. tuberculosis-reactive antibody levels are heterogeneous, studies suggest that levels of IgG glycosylation differ between disease states. Here we extend this observation across antibody domains and M. tuberculosis specificities to define changes with the greatest resolving power. METHODS:Capillary electrophoretic glycan analysis was performed on bulk non-antigen-specific IgG, bulk Fc domain, bulk Fab domain, and purified protein derivative (PPD)- and Ag85A-specific IgG from subjects with latent (n?=?10) and active (n?=?20) tuberculosis. PPD-specific isotype/subclass, PPD-specific antibody-dependent phagocytosis, cellular cytotoxicity, and natural killer cell activation were assessed. Discriminatory potentials of antibody features were evaluated individually and by multivariate analysis. RESULTS:Parallel profiling of whole, Fc, and Fab domain-specific IgG glycosylation pointed to enhanced differential glycosylation on the Fc domain. Differential glycosylation was observed across antigen-specific antibody populations. Multivariate modeling highlighted Fc domain glycan species as the top discriminatory features, with combined PPD IgG titers and Fc domain glycans providing the highest classification accuracy. CONCLUSIONS:Differential glycosylation occurs preferentially on the Fc domain, providing significant discriminatory power between different states of M. tuberculosis infection and disease.
Project description:Immunoglobulin G (IgG), which contains four subclasses (IgG1-4), is one of the most important classes of glycoproteins in the immune system. Because of its importance in the immune system, a steady increase of interest in developing IgG as the biomarker or biotherapeutic agent for the treatment of diseases has been seen, as most therapeutic mAbs were IgG-based. N-Glycosylation of IgG is crucial for its effector function and makes IgG highly heterogeneous both in structure and function, although all four subclasses of IgG contain only a single N-glycosylation site in the Fc region with a highly similar amino acid sequence. Therefore, fine mapping of IgG glycosylation is necessary for understanding the IgG function and avoiding aberrant glycosylation in mAbs. However, site-specific and comprehensive N-glycosylation analysis of IgG subclasses still cannot be achieved by MS alone due to the partial sequence coverage and loss of connections among glycosylation of the protein sequence. We report here a chemical labeling strategy to improve the electron transfer dissociation efficiency in mass spectrometry analysis, which enables a 100% peptide sequence coverage of N-glycopeptides in all subclasses of IgG. Combined with high-energy collisional dissociation for the fragmentation of glycans, fine mapping of the N-glycosylation profile of IgG is achieved. This comprehensive glycosylation analysis strategy for the first time allows the discrimination of IgG3 and IgG4 intact N-glycopeptides with high similarity in sequence without the antibody-based pre-separation. Using this strategy, aberrant serum IgG N-glycosylation for four IgG subclasses associated with cirrhosis and hepatocellular carcinoma was revealed. Moreover, this method identifies 5 times more intact glycopeptides from human serum than the native-ETD method, implying that the approach can also accommodate large-scale site-specific profiling of glycoproteomes.
Project description: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 ?l 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.
Project description:Protein N-glycosylation plays critical roles in controlling brain function, but little is known about human brain N-glycoproteome and its alterations in Alzheimer's disease (AD). Here, we report the first, large-scale, site-specific N-glycoproteome profiling study of human AD and control brains using mass spectrometry-based quantitative N-glycoproteomics. The study provided a system-level view of human brain N-glycoproteins and in vivo N-glycosylation sites and identified disease signatures of altered N-glycopeptides, N-glycoproteins, and N-glycosylation site occupancy in AD. Glycoproteomics-driven network analysis showed 13 modules of co-regulated N-glycopeptides/glycoproteins, 6 of which are associated with AD phenotypes. Our analyses revealed multiple dysregulated N-glycosylation-affected processes and pathways in AD brain, including extracellular matrix dysfunction, neuroinflammation, synaptic dysfunction, cell adhesion alteration, lysosomal dysfunction, endocytic trafficking dysregulation, endoplasmic reticulum dysfunction, and cell signaling dysregulation. Our findings highlight the involvement of N-glycosylation aberrations in AD pathogenesis and provide new molecular and system-level insights for understanding and treating AD.
Project description:Fc? receptors (Fc?R) mediate key functions in immunological responses. For instance, Fc?RIIIa is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Fc?RIIIa interacts with the fragment crystallizable (Fc) of immunoglobulin G (IgG). This interaction is known to be highly dependent on IgG Fc glycosylation. Thus, the impact of glycosylation features on this interaction has been investigated in several studies by numerous analytical and biochemical techniques. Fc?RIIIa affinity chromatography (AC) hyphenated to mass spectrometry (MS) is a powerful tool to address co-occurring Fc glycosylation heterogeneity of monoclonal antibodies (mAbs). However, MS analysis of mAbs at the intact level may provide limited proteoform resolution, for example, when additional heterogeneity is present, such as antigen-binding fragment (Fab) glycosylation. Therefore, we investigated middle-up approaches to remove the Fab and performed AC-MS on the IgG Fc to evaluate its utility for Fc?RIIIa affinity assessment compared to intact IgG analysis. We found the protease Kgp to be particularly suitable for a middle-up Fc?RIIIa AC-MS workflow as demonstrated for the Fab glycosylated cetuximab. The complexity of the mass spectra of Kgp digested cetuximab was significantly reduced compared to the intact level while affinity was fully retained. This enabled a reliable assignment and relative quantitation of Fc glycoforms in Fc?RIIIa AC-MS. In conclusion, our workflow allows a functional separation of differentially glycosylated IgG Fc. Consequently, applicability of Fc?RIIIa AC-MS is extended to Fab glycosylated IgG, i.e., cetuximab, by significantly reducing ambiguities in glycoform assignment vs. intact analysis.
Project description:Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes.
Project description:Immunoglobulin G (IgG) can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may-in addition to affecting antigen binding-contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.
Project description:Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. However, in-depth knowledge of protein glycosylation to uncover functions and their clinical applications requires quantitative glycoproteomics eliciting both peptide and glycan sequences concurrently. Here we describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). LC-MS/MS analysis of de-glycosylated peptides identified 270 mouse serum peptides (95 glycoproteins) as sialylated glycopeptides, of which 67 glycopeptides were fully characterized by MS/MS analyses in a straightforward manner. We revealed the importance of a fragment ion containing innermost N-acetylglucosamine (GlcNAc) residue as MRM transitions regardless the sequence of the peptides. Versatility of the reverse glycoblotting-assisted MRM assays was demonstrated by quantitative comparison of 25 targeted glycopeptides from 16 proteins between mice with homo and hetero types of diabetes disease model.