Mouse milk immunoglobulin G Fc-linked N-glycosylation nano-LC-MS analysis in a model of vancomycin exposure during...

Breast milk immunoglobulin G (IgG) plays an important role in the transfer of passive immunity in early life and in shaping the neonatal immune system through N-glycan-mediated effector functions. Currently, there are no protocols available to analyze breast milk IgG-Fc glycosylation in mouse models. Therefore, we developed and validated a glycoproteomic workflow for the medium-throughput subclass-specific nano-LC-MS analysis of IgG enriched from small milk volumes of lactating mice. With the established methods, the IgG glycopatterns in a mouse model of antibiotic use during pregnancy and increased asthma susceptibility in the offspring were analyzed. Pregnant BALB/c mice were treated with vancomycin during gestation days 8-17 and IgG1F, IgG2, and IgG3-Fc glycosylation was subsequently analyzed in maternal serum, maternal breast milk, and offspring serum on postnatal day 15. The IgG glycosylation profiles of mouse maternal milk and serum revealed no significant differences within the glycoforms quantified across subclasses. However, vancomycin use during pregnancy was associated with changes in IgG-Fc glycosylation in offspring serum, shown by the decreased relative abundance of the IgG1F-G1 and IgG3-G0 glycoforms, together with the increased relative abundance of the IgG3-G2 and S1 glycoforms. The workflow presented will aid in the emerging integrative multi-omics- and glycomics-oriented milk analyses both in rodent models and human cohorts for a better understanding of mother-infant immunological interactions.