Comparative Site-Specific O -Glycosylation Analysis of the Milk Fat Globule Membrane Proteome in Donkey Colostrum and Mature Milk.

Donkey milk fat globule membrane (MFGM) proteins are a class of membrane-bound secreted proteins with broad-spectrum biofunctional activities; however, their site-specific O -glycosylation landscapes have not been systematically mapped. In this study, an in-depth MFGM O -glycoproteome profile of donkey milk during lactation was constructed based on an intact glycopeptide-centered, label-free glycoproteomics pipeline, with 2137 site-specific O -glycans from 1121 MFGM glycoproteins and 619 site-specific O -glycans from 217 MFGM glycoproteins identified in donkey colostrum and donkey mature milk, respectively. As lactation progressed, the number of site-specific O -glycans from three glycoproteins significantly increased, whereas that of 11 site-specific O -glycans from five glycoproteins significantly decreased. Furthermore, donkey MFGM O -glycoproteins with core-1 and core-2 core structures and Lewis and sialylated branch structures may be involved in regulating apoptosis. The findings of this study reveal the differences in the composition of donkey MFGM O -glycoproteins and their site-specific O -glycosylation modification dynamic change rules during lactation, providing a molecular basis for understanding the complexity and biological functions of donkey MFGM protein O -glycosylation.