Glycoproteomic Analysis of Antibodies

Zauner, Gerhild; Selman, Maurice H. J.; Bondt, Albert; Rombouts, Yoann; Blank, Dennis; Deelder, André M.; Wuhrer, Manfred

doi:10.1074/mcp.r112.026005

Cited by 153 publications

(152 citation statements)

References 102 publications

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“…Although it is well demonstrated that correct assembly of the 21 (or 24, depending on the oligomerization status) polypeptides is a prerequisite for the functionality of IgMs (7), the impact of glycosylation is largely unknown. However, results from clinical studies, particularly on IgGs, indicate that carbohydrates play a structural and functional role for all immunoglobulins (8)(9)(10).…”

mentioning

confidence: 99%

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

Loos

Gruber

Altmann

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Significance IgM antibodies are increasingly gaining interest as therapeutics; however, knowledge about this antibody class is scarce. Specifically the impact of N-glycans on the functional mechanism of this heavily glycosylated molecule is entirely unknown. To address this issue we produced different IgM glycoforms in plants and characterized them. Moreover, we present a computer model that explains the characteristic N-glycosylation pattern of IgMs. With the successful in planta generation of recombinant IgMs largely resembling the plasma-derived orthologue, we offer an efficient alternative to mammalian cell-based expression systems. IgMs with targeted glycoengineered N-glycans now enable detailed structure–function studies and will lead to the production of IgMs with optimized in vivo activities.

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mentioning

confidence: 99%

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

Loos

Gruber

Altmann

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Whereas the glycosylation of IgG, IgE and IgA is well studied, there are no detailed mass spectrometric data available describing the site-specific glycosylation profiles of human serum IgM (6 -9). Human IgG has one conserved Nglycosylation site on each heavy chain CH2 domain at Asn 297, and ϳ15-20% of normal polyclonal IgG bears additional Fab (fragment antigen binding) glycosylation (6,10,11). Other antibody classes such as IgM or IgA show a higher complexity with respect to the number of glycosylation sites and variety of glycoforms (6,9).…”

mentioning

confidence: 99%

“…Human IgG has one conserved Nglycosylation site on each heavy chain CH2 domain at Asn 297, and ϳ15-20% of normal polyclonal IgG bears additional Fab (fragment antigen binding) glycosylation (6,10,11). Other antibody classes such as IgM or IgA show a higher complexity with respect to the number of glycosylation sites and variety of glycoforms (6,9). Only recently, also monoclonal IgM antibodies came into the focus of pharmaceutical industry, because they show great potential for the treatment of diseases (12)(13)(14).…”

mentioning

confidence: 99%

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM)*

Pabst

Kuster

Wahl³

et al. 2015

Molecular & Cellular Proteomics

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We demonstrate a new approach for the site-specific identification and characterization of protein N-glycosylation. It is based on a nano-liquid chromatography microarray-matrix assisted laser desorption/ionization-MS platform, which employs droplet microfluidics for onplate nanoliter reactions. A chromatographic separation of a proteolytic digest is deposited at a high frequency on the microarray. In this way, a short separation run is archived into thousands of nanoliter reaction cavities, and chromatographic peaks are spread over multiple array spots. After fractionation, each other spot is treated with PNGaseF to generate two correlated traces within one run, one with treated spots where glycans are enzymatically released from the peptides, and one containing the intact glycopeptides. Mining for distinct glycosites is performed by searching for the predicted deglycosylated peptides in the treated trace. An identified peptide then leads directly to the position of the "intact" glycopeptide clusters, which are located in the adjacent spots. Furthermore, the deglycosylated peptide can be sequenced efficiently in a simple collision-induced dissociation-MS experiment. We applied the microarray approach to a detailed site-specific glycosylation analysis of human serum IgM. By scanning the treated spots with low-resolution matrix assisted laser desorption/ionization-time-offlight-MS, we observed all five deglycosylated peptides, including the one originating from the secretory chain. A detailed glycopeptide characterization was then accomplished on the adjacent, untreated spots with high mass resolution and high mass accuracy using a matrix assisted laser desorption ionization-Fourier transform-MS. We present the first detailed and comprehensive mass spectrometric analysis on the glycopeptide level for human polyclonal IgM with high mass accuracy. Besides complex type glycans on Asn 395, 332, 171, and on the J chain, we observed oligomannosidic glycans on Asn 563, Asn 402 and minor amounts of oligomannosidic glycans on the glycosite Asn 171. Furthermore, hybrid type glycans were found on Asn 402, Asn 171 and in traces Asn 332. Molecular & Cellular

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“…ELLA and impedimetric lectin biosensors. Lectins involved in the study were selected to keep in mind an ability to bind to biantennary complex glycan types often present on human IgG (and other proteins) in human sera at relatively high concentrations and known to be aberrantly glycosylated during a progress of specific (mostly autoimmune) diseases [46]. Antibody against BSA was selected as a glycoprotein to perform calibration of lectin microarray technique with eight different lectins.…”

Section: Selection Of Lectinsmentioning

confidence: 99%

Glycoprofiling as a novel tool in serological assays of systemic sclerosis: A comparative study with three bioanalytical methods

Kluková

Bertók

Petrikova

et al. 2015

Analytica Chimica Acta

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Systemic sclerosis (SSc) is an autoimmune disease seriously affecting patient´s quality of life. The heterogeneity of the disease also means that identification and subsequent validation of biomarkers of the disease is quite challenging. A fully validated single biomarker for diagnosis, prognosis, disease activity and assessment of response to therapy is not yet available. The main aim of this study was to apply an alternative assay protocol to the immunoassay-based analysis of this disease by employment of sialic acid recognizing lectin Sambucus nigra agglutinin (SNA) to glycoprofile serum samples. To our best knowledge this is the first study describing direct lectin-based glycoprofiling of serum SSc samples. Three different analytical methods for glycoprofiling of serum samples relying on application of lectins are compared here from a bioanalytical point of view including traditional ELISA-like lectin-based method (ELLA), novel fluorescent lectin microarrays and ultrasensitive impedimetric lectin biosensors. Results obtained by all three bioanalytical methods consistently showed differences in the level of sialic acid present on glycoproteins, when serum from healthy people was compared to the one from patients having SSc. Thus, analysis of sialic acid content in human serum could be of a diagnostic value for future detection of SSc, but further work is needed to enhance selectivity of assays for example by glycoprofiling of a fraction of human serum enriched in antibodies for individual diagnostics.

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Glycoproteomic Analysis of Antibodies

Cited by 153 publications

References 102 publications

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM)*

Glycoprofiling as a novel tool in serological assays of systemic sclerosis: A comparative study with three bioanalytical methods

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