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High-Throughput Analysis of IgG Fc Glycopeptides by LC-MS

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High-Throughput Glycomics and Glycoproteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1503))

Abstract

This chapter contains a nanoscale liquid chromatography–mass spectrometry method for the glycoform profiling of the conserved Fc N-glycosylation site of monoclonal and polyclonal immunoglobulin G (IgG). It describes in detail LaCyTools, a program for automated data (pre-)processing of the obtained LC-MS data. The minimal sample preparation necessary is explained as well as an optional method for affinity purification of (polyclonal) antibodies from serum or plasma.

After (optional) affinity purification, the pure IgG is cleaved with trypsin. The tryptic glycopeptides are separated almost exclusively on their peptide backbone. This ensures similar response factors for all glycoforms in the MS detection and allows the collection of separate glycoform profiles for different IgG isoforms or allotypes. LaCyTools automatically performs label-free (relative) quantitation of the obtained data after minimal manual input and additionally calculates several quality criteria which can be used for data curation at the level of both individual analytes and entire LC-MS runs.

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References

  1. Beck A, Wagner-Rousset E, Ayoub D, et al. (2013) Characterization of therapeutic antibodies and related products. Anal Chem 85:715–736

    Article  CAS  PubMed  Google Scholar 

  2. Dotz V, Haselberg R, Shubhakar A, et al. (2015) Mass spectrometry for glycosylation analysis of biopharmaceuticals. Trends Anal Chem 73:1–9

    Article  CAS  Google Scholar 

  3. Schwab I, Nimmerjahn F (2014) Role of sialylation in the anti-inflammatory activity of intravenous immunoglobulin—F(ab′)(2) versus Fc sialylation. Clin Exp Immunol 178(Suppl 1):97–99

    Article  PubMed  PubMed Central  Google Scholar 

  4. Bondt A, Rombouts Y, Selman MH, et al. (2014) Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes. Mol Cell Proteomics 13:3029–3039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Jones AJ, Papac DI, Chin EH, et al. (2007) Selective clearance of glycoforms of a complex glycoprotein pharmaceutical caused by terminal N-acetylglucosamine is similar in humans and cynomolgus monkeys. Glycobiology 17:529–540

    Article  CAS  PubMed  Google Scholar 

  6. Mimura Y, Sondermann P, Ghirlando R, et al. (2001) Role of oligosaccharide residues of IgG1-Fc in Fc gamma RIIb binding. J Biol Chem 276:45539–45547

    Article  CAS  PubMed  Google Scholar 

  7. Sesarman A, Vidarsson G, Sitaru C (2010) The neonatal Fc receptor as therapeutic target in IgG-mediated autoimmune diseases. Cell Mol Life Sci 67:2533–2550

    Article  CAS  PubMed  Google Scholar 

  8. Reusch D, Haberger M, Falck D, et al. (2015) Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles-Part 2: mass spectrometric methods. MAbs 7:732–742

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Vidarsson G, Dekkers G, Rispens T (2014) IgG subclasses and allotypes: from structure to effector functions. Front Immunol 5:520

    Article  PubMed  PubMed Central  Google Scholar 

  10. Bruker Daltonics. https://www.bruker.com/products/mass-spectrometry-and-separations/ms-software/compass/overview.html. Accessed 11 Mar 2015

  11. Schilling B, Rardin MJ, MacLean BX, et al. (2012) Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation. Mol Cell Proteomics 11:202–214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jansen BC, Reiding KR, Bondt A, et al. (2015) MassyTools: a high throughput targeted data processing tool for relative quantitation and quality control developed for glycomic and glycoproteomic MALDI-MS. J Proteome Res 14:5088–5098

    Article  CAS  PubMed  Google Scholar 

  13. Van Rossum G, Drake FL Jr (1995) Python reference manual. Centrum voor Wiskunde en Informatica, Amsterdam

    Google Scholar 

  14. Van Der Walt S, Colbert SC, Varoquaux G (2011) The NumPy array: a structure for efficient numerical computation. Comput Sci Eng 13:22–30

    Article  Google Scholar 

  15. Oliphant TE (2007) Python for scientific computing. Comput Sci Eng 9:10–20

    Article  CAS  Google Scholar 

  16. Hunter JD (2007) Matplotlib: A 2D graphics environment. Comput Sci Eng 9:90–95

    Article  Google Scholar 

  17. Python(x,y). http://python-xy.github.io/. Accessed 21 Aug 2015

  18. Anaconda. https://www.continuum.io/downloads. Accessed 14 July 2015

  19. Falck D, Jansen BC, Plomp R, et al. (2015) 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

    Article  CAS  PubMed  Google Scholar 

  20. Switzar L, Giera M, Niessen WM (2013) Protein digestion: an overview of the available techniques and recent developments. J Proteome Res 12:1067–1077

    Article  CAS  PubMed  Google Scholar 

  21. Hustoft HK, Reubsaet L, Greibrokk T, et al. (2011) Critical assessment of accelerating trypsination methods. J Pharm Biomed Anal 56:1069–1078

    Article  CAS  PubMed  Google Scholar 

  22. Rombouts Y, Ewing E, van de Stadt LA, et al. (2015) Anti-citrullinated protein antibodies acquire a pro-inflammatory Fc glycosylation phenotype prior to the onset of rheumatoid arthritis. Ann Rheum Dis 74:234–241

    Article  CAS  PubMed  Google Scholar 

  23. Kessner D, Chambers M, Burke R, et al. (2008) ProteoWizard: open source software for rapid proteomics tools development. Bioinformatics 24:2534–2536

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pluskal T, Castillo S, Villar-Briones A, et al. (2010) MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Biochem 11:395

    Google Scholar 

  25. Strohalm M, Kavan D, Novák P, et al. (2010) mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data. Anal Chem 82:4648–4651

    Article  CAS  PubMed  Google Scholar 

  26. Geany. http://www.geany.org/. Accessed 15 Oct 2015

  27. IBM SPSS Statistics. http://www-01.ibm.com/software/analytics/spss/products/statistics/. Accessed 3 Nov 2015

  28. Microsoft Excel. https://products.office.com/en-us/excel. Accessed 3 Nov 2015

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Author information

Authors and Affiliations

  1. Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333, ZA, Leiden, The Netherlands

    David Falck, Bas C. Jansen, Noortje de Haan & Manfred Wuhrer

  2. Division of BioAnalytical Chemistry, VU University Amsterdam, De Boelelaan 1083, 1081, HV, Amsterdam, The Netherlands

    Manfred Wuhrer

Authors
  1. David Falck
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  2. Bas C. Jansen
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  3. Noortje de Haan
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  4. Manfred Wuhrer
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Corresponding author

Correspondence to Manfred Wuhrer .

Editor information

Editors and Affiliations

  1. Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia

    Gordan Lauc

  2. Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

    Manfred Wuhrer

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Falck, D., Jansen, B.C., de Haan, N., Wuhrer, M. (2017). High-Throughput Analysis of IgG Fc Glycopeptides by LC-MS. In: Lauc, G., Wuhrer, M. (eds) High-Throughput Glycomics and Glycoproteomics. Methods in Molecular Biology, vol 1503. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6493-2_4

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  • DOI: https://doi.org/10.1007/978-1-4939-6493-2_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6491-8

  • Online ISBN: 978-1-4939-6493-2

  • eBook Packages: Springer Protocols

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