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

Area of Science:

• Immunology
• Glycobiology
• Analytical Chemistry

Background:

• Fc gamma receptor IIIa (FcγRIIIa/CD16a) regulates IgG effector functions, with dysregulation linked to inflammatory diseases.
• Specific glycoforms of FcγRIIIa, particularly those with high-mannose N-linked glycans at site N162, exhibit higher affinity for IgG.
• Understanding FcγRIIIa glycosylation is crucial for its role in immunity, immunotherapy, and immune disorders.

Purpose of the Study:

• To develop and validate a targeted nanoliquid chromatography tandem mass spectrometry parallel reaction monitoring (PRM) method for site-specific analysis of FcγRIIIa N-glycosylation.
• To investigate changes in FcγRIIIa N-glycosylation at site N162 during monocyte differentiation.
• To assess FcγRIIIa N-glycosylation in primary human immune cells.

Main Methods:

• Development of a targeted nanoliquid chromatography tandem mass spectrometry parallel reaction monitoring (PRM) assay.
• Application of the PRM method to analyze FcγRIIIa N-glycosylation site N162.
• Analysis of FcγRIIIa from a monocyte differentiation model and primary human monocytes/peripheral blood mononuclear cells.

Main Results:

• The developed PRM method enables site-specific study of FcγRIIIa N-glycosylation.
• FcγRIIIa N-glycosylation at site N162 was consistent across different cell types analyzed.
• An increase in high-mannose N-glycan structures at site N162 was observed during monocyte differentiation, correlating with high-affinity FcγRIIIa glycoforms.

Conclusions:

• Targeted glycopeptide PRM is a feasible strategy for studying FcγRIIIa glycosylation in primary cells.
• The findings demonstrate dynamic changes in FcγRIIIa glycosylation during monocyte differentiation.
• This analytical approach is adaptable for clinical studies investigating FcγRIIIa glycosylation in health and disease.