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Absolute Quantitation of Glycoforms of Two Human IgG Subclasses Using Synthetic Fc Peptides and Glycopeptides

  • Rini Roy
  • Evelyn Ang
  • Emy Komatsu
  • Ronald Domalaon
  • Adrien Bosseboeuf
  • Jean Harb
  • Sylvie Hermouet
  • Oleg Krokhin
  • Frank Schweizer
  • Hélène Perreault
Focus: Mass Spectrometry in Glycobiology and Related Fields: Research Article

Abstract

Immunoglobulins, such as immunoglobulin G (IgG), are of prime importance in the immune system. Polyclonal human IgG comprises four subclasses, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitative method for these subclasses and their Fc N-glycoforms, (glyco)peptides were synthesized using a solid-phase approach and used as internal standards. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR standards, respectively. For IgG1, a similar glycopeptide where tyrosine (Y) was isotopically labelled was used to quantify monoclonal IgG1 that had been treated with the enzyme Endo-F2, i.e., yielding tryptic glycopeptide EEQYN(GlcNAc)STYR. The next step was to quantify single subclasses within polyclonal human IgG samples. Although ion abundances in the MALDI spectra often showed higher signals for IgG2 than IgG1, depending on the spotting solvent used, determination of amounts using the newly developed quantitative method allowed to obtain accurate concentrations where IgG1 species were predominant. It was observed that simultaneous analysis of IgG1 and IgG2 yielded non-quantitative results and that more success was obtained when subclasses were quantified one by one. More experiments served to assess the respective extraction and ionization efficiencies of EEQYNSTYR/EEQFNSTFR and EEQYN(GlcNAc)STYR/EEQFN(GlcNAc)STFR mixtures under different solvent and concentration conditions.

Graphical Abstract

Keywords

Immunoglobulins Glycopeptides MALDI-MS Quantitative analysis Internal standard Glycoproteomics MALDI-ToF-MS Absolute quantitation IgG tryptic digests 

Notes

Acknowledgments

The authors would like to thank James Rini (University of Toronto) for providing the Her2SF mAb sample, as well as Michael Butler and Carina Villacrès for providing the Eg2-hFc mAb.

Funding Information

This study was funded by the Natural Sciences and Engineering Council of Canada (NSERC) and the Canadian Foundation for Innovation (CFI).

Supplementary material

13361_2018_1900_MOESM1_ESM.docx (821 kb)
ESM 1 (DOCX 821 kb)

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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of ManitobaWinnipegCanada
  2. 2.Manitoba Centre for Proteomics and Systems BiologyUniversity of ManitobaWinnipegCanada
  3. 3.CRCINA, Inserm U1232, Institut de Recherche en Santé 2Université de NantesNantesFrance
  4. 4.Centre de Recherche en Transplantation et Immunologie UMR1064, InsermUniversité de NantesNantesFrance

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