Site-Specific N‑Glycoprofiling of Immunoglobulin G Subtypes from Donkey Milk

Donkey milk IgG was probed for the site-specific N-glycosylation pattern through RP-UHPLC-MS/MS. The affinity-purified milk IgG was subjected to SDS-PAGE and proteomic analysis, which revealed the presence of subtypes. Multiple N-glycopeptides arising from the predicted donkey IgG1, IgG2, IgG3, IgG5, IgG6, and IgG7 subtypes' heavy-chain constant region were shown to contain glycans at the highly conserved glycosylation site NST in the CH2 domain. Differences in the peptide backbone with the NST site among subtypes generated after trypsin digestion resulted in the evaluation of the subtype-specific glycan pattern. Glycan sequence analysis indicated predominantly biantennary complex types with core fucosylation at the site NST. Interestingly, an additional site NQT in the CH1 domain of the heavy-chain constant region of IgG5 was found to possess mainly sialylated biantennary complex glycans with NeuAc and NeuGc. Structural diversity of glycans was mainly observed in the predicted donkey IgG1, IgG5, and IgG7, whereas IgG2, IgG3, and IgG6 resulted in the glycopeptides that are of low abundance in the analyzed samples. These findings would pave the way for a better understanding of donkey milk functional properties.