A novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on human IgG
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ORIGINAL ARTICLE
A novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on human IgG Osmond D. Rebello 1,2 & Richard A. Gardner 1 & Paulina A. Urbanowicz 1 & David N. Bolam 3 & Lucy I. Crouch 4 & David Falck 2 & Daniel I. R. Spencer 1 Received: 8 August 2020 / Revised: 28 September 2020 / Accepted: 6 October 2020 # The Author(s) 2020
Abstract Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLDMSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson’s r correlation coefficient of 0.8208 between the two methods. Keywords Glycomics . Plate assay . HPLC-FLD-MS . Glycosidase . Glycans . Galactosylation . Sialylation . Antibodies
Introduction Changes in IgG glycosylation have been associated with certain inflammatory diseases such as rheumatoid arthritis [1, 2], systemic lupus erythematosus [3], type 2 diabetes [4] and Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10719-020-09953-9) contains supplementary material, which is available to authorized users. * Osmond D. Rebello [email protected] * Daniel I. R. Spencer [email protected] 1
Ludger Ltd, Culham Science Centre, Abingdon, UK
2
Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands
3
Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
4
Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK
inflammatory bowel disease (IBD) [5]. During an inflammatory state, a loss of systemic and/or cellular homeostasis may trigger a change in the glycosylation of IgG molecules [6, 7]. T
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