Assessing High-Affinity N -Glycoforms of the Human Fcγ Receptor IIIa (CD16a) via Parallel Reaction Monitoring Mass Spectrometry

The Fc γ-receptor IIIa (FcγRIIIa), or CD16a, is a receptor for the Fc region of IgG that plays an essential role in the regulation of antibody effector functions. Dysregulated CD16a activity contributes to tissue damage and chronic inflammation or, conversely, compromises the removal of immune complexes. CD16a glycoforms with high-mannose N-linked glycans at site N162 demonstrate higher affinity for IgG compared to other forms. Here, we developed a targeted nanoliquid chromatography tandem mass spectrometry parallel reaction monitoring (PRM) method for the site-specific study of CD16a N-glycosylation. The method was applied to assess changes in site N162 N-glycosylation in a model of monocyte differentiation and in CD16a from human primary monocytes and peripheral blood mononuclear cells. Site N162 N-glycosylation was similar across the surveyed cell types. An increase in the proportion of high-mannose N-glycan compositions at site N162 was observed, corresponding to high-affinity glycoforms of CD16a, in a model of monocyte differentiation. Here, the feasibility of using targeted glycopeptide PRM to study CD16a glycosylation in primary cells has been demonstrated. This strategy is well-suited for adaptation to clinical studies focused on the role of CD16a glycosylation in physiology, immunotherapy, and immune disorders. Data are available via ProteomeXchange with the identifier PXD071784.