Proteomic, N-glycoproteomic, and phosphoproteomic profiling reveals dynamic remodeling of goat milk fat globule membrane proteins across lactational stages.

Goat milk fat globule membrane (MFGM) proteins are critical for early development, immune regulation, and metabolic homeostasis in neonatal offspring. However, the composition of MFGM proteins in goat colostrum (GC) and mature milk (GM) and the dynamic alterations in their N-glycosylation and phosphorylation have yet to be elucidated. To comprehensively characterize the dynamic alterations of MFGM proteins between GC and GM, this study employed Astral-data-independent acquisition proteomics, phosphoproteomics, and label-free quantitative N-glycoproteomics. A total of 3,129 proteins, 67 N-glycosylated, and 1,016 phosphorylated proteins were identified. Intergroup differential analysis revealed no significant differences at the protein level, and only 4 differential N-glycosylated peptides were identified (FDR <0.05). In contrast, the phosphoproteome exhibited robust alterations, retaining 201 high-confidence differentially phosphorylated proteins. During the GC phase, the highly expressed MFGM proteins and their modified forms were mainly involved in intestinal IgA synthesis, complement and coagulation cascades, as well as metabolic pathways for riboflavin, tryptophan, and vitamin B6. Further protein-protein interaction analysis identified core hub proteins and key functional modules linking immunity, lipid metabolism, and material transport. Collectively, our findings suggest that the functional remodeling of goat MFGM across early lactation may be largely driven by dynamic protein phosphorylation, rather than changes in protein abundance or N-glycosylation occupancy itself, and this process may contribute to neonatal passive immune development and improved digestive functionality. These new insights provide an important basis for optimizing early rearing strategies and developing milk replacers for goats.