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GlYcoLISA: antigen-specific and subclass-specific IgG Fc glycosylation analysis based on an immunosorbent assay with an LC–MS readout

Nature Protocols (2024)Cite this article

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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

  • This protocol describes a method for profiling fragment crystallizable glycosylation of antigen-specific antibodies isolated from clinical samples.

  • 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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Fig. 1: IgG structure and expected glycosylation structures.
Fig. 2: GlYcoLISA workflow.
Fig. 3: Representative mass spectra.
Fig. 4: Data structure and processing.
Fig. 5: Intermediate precision of the method.
Fig. 6: Anti-S IgG Fc glycosylation dynamically changes over the course of SARS-CoV-2 infection.
Fig. 7: High anti-HLA galactosylation and sialylation are associated with the immunological destruction of transfused platelets in patients with chemotherapy-induced thrombocytopenia.

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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.

References

  1. Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M. & Dwek, R. A. The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu. Rev. Immunol. 25, 21–50 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. de Haan, N., Falck, D. & Wuhrer, M. Monitoring of immunoglobulin N- and O-glycosylation in health and disease. Glycobiology 30, 226–240 (2020).

    Article  PubMed  Google Scholar 

  3. Ferrara, C. et al. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose. Proc. Natl Acad. Sci. USA 108, 12669–12674 (2011).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. van Osch, T. L. J. et al. Fc galactosylation promotes hexamerization of human IgG1, leading to enhanced classical complement activation. J. Immunol. 207, 1545–1554 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Kapur, R., Einarsdottir, H. K. & Vidarsson, G. IgG-effector functions: ‘the good, the bad and the ugly’. Immunol. Lett. 160, 139–144 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Shields, R. L. et al. Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity. J. Biol. Chem. 277, 26733–26740 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Shibata-Koyama, M. et al. Nonfucosylated rituximab potentiates human neutrophil phagocytosis through its high binding for FcγRIIIb and MHC class II expression on the phagocytotic neutrophils. Exp. Hematol. 37, 309–321 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Beck, A. & Reichert, J. M. Marketing approval of mogamulizumab: a triumph for glyco-engineering. MAbs 4, 419–425 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Dekkers, G. et al. Decoding the human immunoglobulin G-glycan repertoire reveals a spectrum of Fc-receptor- and complement-mediated-effector activities. Front. Immunol. 8, 877 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Vergroesen, R. D. et al. B-cell receptor sequencing of anti-citrullinated protein antibody (ACPA) IgG-expressing B cells indicates a selective advantage for the introduction of N-glycosylation sites during somatic hypermutation. Ann. Rheum. Dis. 77, 956–958 (2018).

    PubMed  Google Scholar 

  11. Trbojevic-Akmacic, I., Abdel-Mohsen, M., Falck, D. & Rapp, E. Editorial: immunoglobulin glycosylation analysis: state-of-the-art methods and applications in immunology. Front. Immunol. 13, 923393 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bondt, A. et al. ACPA IgG galactosylation associates with disease activity in pregnant patients with rheumatoid arthritis. Ann. Rheum. Dis. 77, 1130–1136 (2018).

    CAS  PubMed  Google Scholar 

  13. Larsen, M. D. et al. Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity. Science 371, eabc8378 (2021).

    Article  CAS  PubMed  Google Scholar 

  14. Kapur, R. et al. A prominent lack of IgG1–Fc fucosylation of platelet alloantibodies in pregnancy. Blood 123, 471–480 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bartsch, Y. C. et al. IgG Fc sialylation is regulated during the germinal center reaction following immunization with different adjuvants. J. Allergy Clin. Immunol. 146, 652–666 e611 (2020).

    Article  CAS  PubMed  Google Scholar 

  16. Wojcik, I. et al. A functional spleen contributes to afucosylated IgG in humans. Sci. Rep. 11, 24045 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Cao, Y. et al. Cytokines in the immune microenvironment change the glycosylation of IgG by regulating intracellular glycosyltransferases. Front. Immunol. 12, 724379 (2021).

    Article  CAS  PubMed  Google Scholar 

  18. Larsen, M. D. et al. Afucosylated Plasmodium falciparum-specific IgG is induced by infection but not by subunit vaccination. Nat. Commun. 12, 5838 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Buhre, J. S. et al. mRNA vaccines against SARS-CoV-2 induce comparably low long-term IgG Fc galactosylation and sialylation levels but increasing long-term IgG4 responses compared to an adenovirus-based vaccine. Front. Immunol. 13, 1020844 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Pongracz, T. et al. Immunoglobulin G1 Fc glycosylation as an early hallmark of severe COVID-19. EBioMedicine 78, 103957 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kemna, M. J. et al. Galactosylation and sialylation levels of IgG predict relapse in patients with PR3–ANCA-associated vasculitis. EBioMedicine 17, 108–118 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Wang, J. et al. Fc-glycosylation of IgG1 is modulated by B-cell stimuli. Mol. Cell. Proteom. 10, 004655 (2011).

    Article  Google Scholar 

  23. Ercan, A. et al. Estrogens regulate glycosylation of IgG in women and men. JCI Insight 2, e89703 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Washburn, N. et al. Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity. Proc. Natl Acad. Sci. USA 112, E1297–E1306 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Falck, D., Jansen, B. C., de Haan, N. & Wuhrer, M. High-throughput analysis of IgG Fc glycopeptides by LC–MS. Methods Mol. Biol. 1503, 31–47 (2017).

    Article  CAS  PubMed  Google Scholar 

  26. Wuhrer, M. et al. Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum. Proteomics 7, 4070–4081 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Selman, M. H. et al. Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC–MS using a sheath-flow ESI sprayer interface. J. Proteom. 75, 1318–1329 (2012).

    Article  CAS  Google Scholar 

  28. Kammeijer, G. S. et al. Dopant enriched nitrogen gas combined with sheathless capillary electrophoresis-electrospray ionization-mass spectrometry for improved sensitivity and repeatability in glycopeptide analysis. Anal. Chem. 88, 5849–5856 (2016).

    Article  CAS  PubMed  Google Scholar 

  29. Falck, D. et al. Glycoforms of immunoglobulin G based biopharmaceuticals are differentially cleaved by trypsin due to the glycoform influence on higher-order structure. J. Proteome Res. 14, 4019–4028 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Simurina, M. et al. Glycosylation of immunoglobulin G associates with clinical features of inflammatory bowel diseases. Gastroenterology 154, e1310 (2018).

    Article  Google Scholar 

  31. Jansen, B. C. et al. LaCyTools: a targeted liquid chromatography–mass spectrometry data processing package for relative quantitation of glycopeptides. J. Proteome Res. 15, 2198–2210 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Falck, D. et al. Glycoform-resolved pharmacokinetic studies in a rat model employing glycoengineered variants of a therapeutic monoclonal antibody. MAbs 13, 1865596 (2021).

    Article  PubMed  Google Scholar 

  33. Scherer, H. U. et al. Immunoglobulin 1 (IgG1) Fc-glycosylation profiling of anti-citrullinated peptide antibodies from human serum. Proteom. Clin. Appl. 3, 106–115 (2009).

    Article  CAS  Google Scholar 

  34. Rombouts, Y. et al. Anti-citrullinated protein antibodies acquire a pro-inflammatory Fc glycosylation phenotype prior to the onset of rheumatoid arthritis. Ann. Rheum. Dis. 74, 234–241 (2015).

    Article  CAS  PubMed  Google Scholar 

  35. Wuhrer, M. et al. Skewed Fc glycosylation profiles of anti-proteinase 3 immunoglobulin G1 autoantibodies from granulomatosis with polyangiitis patients show low levels of bisection, galactosylation, and sialylation. J. Proteome Res. 14, 1657–1665 (2015).

    Article  CAS  PubMed  Google Scholar 

  36. Sonneveld, M. E. et al. Patients with IgG1-anti-red blood cell autoantibodies show aberrant Fc-glycosylation. Sci. Rep. 7, 8187 (2017).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  37. Kapur, R. et al. Low anti-RhD IgG-Fc-fucosylation in pregnancy: a new variable predicting severity in haemolytic disease of the fetus and newborn. Br. J. Haematol. 166, 936–945 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Sonneveld, M. E. et al. Antigen specificity determines anti-red blood cell IgG-Fc alloantibody glycosylation and thereby severity of haemolytic disease of the fetus and newborn. Br. J. Haematol. 176, 651–660 (2017).

    Article  CAS  PubMed  Google Scholar 

  39. Sonneveld, M. E. et al. Fc-glycosylation in human IgG1 and IgG3 is similar for both total and anti-red-blood cell anti-K antibodies. Front. Immunol. 9, 129 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Siekman, S. L. et al. The IgG glycome of SARS-CoV-2 infected individuals reflects disease course and severity. Front. Immunol. 13, 993354 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ackerman, M. E. et al. Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity. J. Clin. Invest. 123, 2183–2192 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lu, L. L. et al. Antibody Fc glycosylation discriminates between latent and active tuberculosis. J. Infect. Dis. 222, 2093–2102 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Van Coillie, J. et al. The BNT162b2 mRNA SARS-CoV-2 vaccine induces transient afucosylated IgG1 in naive but not in antigen-experienced vaccinees. EBioMedicine 87, 104408 (2023).

    Article  PubMed  Google Scholar 

  44. van Osch, T. L. J. et al. Altered Fc glycosylation of anti-HLA alloantibodies in hemato-oncological patients receiving platelet transfusions. J. Thromb. Haemost. 20, 3011–3025 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Bharadwaj, P. et al. Afucosylation of HLA-specific IgG1 as a potential predictor of antibody pathogenicity in kidney transplantation. Cell Rep. Med. 3, 100818 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Sokolova, M. V. et al. Antibodies against citrullinated proteins of IgA isotype are associated with progression to rheumatoid arthritis in individuals at-risk. RMD Open. 9, e002705 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Lai, K. N. et al. IgA nephropathy. Nat. Rev. Dis. Prim. 2, 16001 (2016).

    Article  PubMed  Google Scholar 

  48. Steffen, U. et al. IgA subclasses have different effector functions associated with distinct glycosylation profiles. Nat. Commun. 11, 120 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  49. Momcilovic, A. et al. Simultaneous immunoglobulin A and G glycopeptide profiling for high-throughput applications. Anal. Chem. 92, 4518–4526 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Chandler, K. B. et al. Multi-isotype glycoproteomic characterization of serum antibody heavy chains reveals isotype- and subclass-specific N-glycosylation profiles. Mol. Cell. Proteom. 18, 686–703 (2019).

    Article  CAS  Google Scholar 

  51. Overall, M. L., Marzuki, S. & Hertzog, P. J. Comparison of different ELISAs for the detection of monoclonal antibodies to human interferon-α. Implications for antibody screening. J. Immunol. Methods 119, 27–33 (1989).

    Article  CAS  PubMed  Google Scholar 

  52. Yuan, S. et al. Changes in anti-thyroglobulin IgG glycosylation patterns in Hashimoto’s thyroiditis patients. J. Clin. Endocrinol. Metab. 100, 717–724 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. Sahoo, A. et al. A clade C HIV-1 vaccine protects against heterologous SHIV infection by modulating IgG glycosylation and T helper response in macaques. Sci. Immunol. 7, eabl4102 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Aoyama, M. et al. Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies. MAbs 11, 826–836 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Scherer, H. U. et al. Glycan profiling of anti-citrullinated protein antibodies isolated from human serum and synovial fluid. Arthritis Rheum. 62, 1620–1629 (2010).

    Article  CAS  PubMed  Google Scholar 

  56. Amez Martin, M., Wuhrer, M. & Falck, D. Serum and plasma immunoglobulin G Fc N-glycosylation is stable during Storage. J. Proteome Res. 20, 2935–2941 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Amez-Martin, M., Wuhrer, M. & Falck, D. Immunoglobulin G glycoprofiles are unaffected by common bottom-up sample processing. J. Proteome Res. 19, 4158–4162 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. de Haan, N. et al. Developments and perspectives in high-throughput protein glycomics: enabling the analysis of thousands of samples. Glycobiology 32, 651–663 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Thermo Fisher Scientific Inc. Chromatography Troubleshooting Guides—Liquid Chromatography https://www.thermofisher.com/nl/en/home/industrial/chromatography/chromatography-learning-center/chromatography-consumables-resources/chromatography-troubleshooting-guides/chromatography-troubleshooting-guides-liquid-chromatography.html (2006).

  60. Pongracz, T., Vidarsson, G. & Wuhrer, M. Antibody glycosylation in COVID-19. Glycoconj. J. 39, 335–344 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Vattepu, R., Sneed, S. L. & Anthony, R. M. Sialylation as an important regulator of antibody function. Front. Immunol. 13, 818736 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Bartsch, Y. C. et al. Sialylated autoantigen-reactive IgG antibodies attenuate disease development in autoimmune mouse models of lupus nephritis and rheumatoid arthritis. Front. Immunol. 9, 1183 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Sustic, T. et al. Immunoassay for quantification of antigen-specific IgG fucosylation. EBioMedicine 81, 104109 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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  1. Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, the Netherlands

    David Falck & Manfred Wuhrer

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D.F. wrote and M.W. corrected the manuscript.

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Correspondence to David Falck or Manfred Wuhrer.

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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

Extended data

Extended Data Table 1 Compositions, nomenclature and structure
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Supplementary information

Source data

Source Data Fig. 5

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 (2024). https://doi.org/10.1038/s41596-024-00963-7

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