Abstract
Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation modulates effector functions such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Consequently, assessing IgG Fc glycosylation is important for understanding the role of antibodies in infectious, alloimmune and autoimmune diseases. GlYcoLISA determines the Fc glycosylation of antigen-specific IgG by an immunosorbent assay with a liquid chromatography–mass spectrometry (LC–MS) readout. Detection of antigen-specific IgG glycosylation in a subclass- and site-specific manner is realized by LC–MS-based glycopeptide analysis after proteolytic cleavage. GlYcoLISA addresses challenges related to the low abundance of specific IgG and the high background of total IgG by using well-established immunosorbent assays for purifying antibodies of the desired specificity using immobilized antigen. Alternative methods with sufficient glycan resolution lack these important specificities. GlYcoLISA is performed in a 96-well plate format, and the analysis of 160 samples takes ~5 d, with 1 d for sample preparation, 2 d of LC–MS measurement and 2 d for partially automated data processing. GlYcoLISA requires expertise in LC–MS operation and data processing.
Key points
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This protocol describes a method for profiling fragment crystallizable glycosylation of antigen-specific antibodies isolated from clinical samples.
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By characterizing IgG specific for an antigen of interest and at a high molecular resolution, the technique allows more confident and functionally relevant characterization of glycosylation alterations in disease than could be achieved with less-specific methods.
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Data availability
All data reported here (Figs. 3 and 5–7) have been previously reported elsewhere or are available in the source data: Fig. 3 and ref. 60 are available upon request from t.pongracz@lumc.nl; Fig. 5, source data; Fig. 6, ref. 20 and Supplementary Table 1; and Fig. 7, ref. 44 and Supplementary Table 1. Source data are provided with this paper.
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Acknowledgements
We acknowledge support from the European Union (European Research Council Synergy, GlycanSwitch, Project 101071386) and the Dutch Research Council in the framework of the Domain Science (ENW) public–private partnership (PPP) Fund for the top sectors (Proteoform-resolved pharmacokinetics of biopharmaceuticals, project no. 019.012), with co-funding through the PPP Allowance made available by Health–Holland, Top Sector Life Sciences & Health. We thank M. Ceschi for the creating of the GlYcoLISA logo (included in Fig. 2).
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Key references in the development of the protocol
Larsen, M. D. et al. Science 371, eabc8378 (2021): https://doi.org/10.1126/science.abc8378
Larsen, M. D. et al. Nat. Commun. 12, 5838 (2021): https://doi.org/10.1038/s41467-021-26118-w
Jansen, B. C. et al. J. Proteome Res. 15, 2198–2210 (2016): https://doi.org/10.1021/acs.jproteome.6b00171
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Relative abundances of glycans and glycosylation traits underlying Fig. 5.
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Falck, D., Wuhrer, M. GlYcoLISA: antigen-specific and subclass-specific IgG Fc glycosylation analysis based on an immunosorbent assay with an LC–MS readout. Nat Protoc 19, 1887–1909 (2024). https://doi.org/10.1038/s41596-024-00963-7
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DOI: https://doi.org/10.1038/s41596-024-00963-7
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