Site-Specific Glycosylation Mapping of Fc Gamma Receptor IIIb from Neutrophils of Individual Healthy Donors

Iwona Wojcik, Thomas Sénard, Erik L de Graaf, George M C Janssen, Arnoud H de Ru, Yassene Mohammed, Peter A van Veelen, Gestur Vidarsson, Manfred Wuhrer, David Falck

Research output: Contribution to journalArticleAcademicpeer-review


Fc gamma receptors (FcγRs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and FcγR impacts their interaction dramatically. Regarding FcγR glycosylation profiling, major analytical challenges are associated with the presence of multiple glycosylation sites in close proximity and large structural heterogeneity. To address these challenges, we developed a straightforward and comprehensive analytical methodology to map FcγRIIIb glycosylation in primary human cells. After neutrophil isolation and immunoprecipitation, glycopeptides containing a single site each were generated by a dual-protease in-gel digestion. The complex mixture was resolved by liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing information on the level of individual donors. In contrast to recently published alternatives for FcγRIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying FcγRIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous FcγRIIIa on natural killer cells. This method will allow assessment of differences in FcγRIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions.

Original languageEnglish
Pages (from-to)13172-13181
Number of pages10
JournalAnalytical Chemistry
Issue number19
Publication statusPublished - 6 Oct 2020


  • Chromatography, Liquid
  • GPI-Linked Proteins/analysis
  • Glycosylation
  • Healthy Volunteers
  • Humans
  • Neutrophils/chemistry
  • Protein Interaction Mapping
  • Receptors, IgG/analysis
  • Tandem Mass Spectrometry


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