Astral-DIA proteomics and 4D label-free N-glycoproteomics of milk fat globule membrane proteins reveal distinct profiles between yak colostrum and mature milk

Milk fat globule membrane (MFGM) proteins are highly glycosylated and participate in various biological processes. However, the N-glycoproteomic profiles of MFGM protein in yak colostrum (YC) and mature milk (YM) remain unexplored. In this study, Astral data-independent acquisition proteomics was applied to analyze the MFGM proteomes of YC and YM, while 4D label-free quantification was used to investigate their N-glycoproteomes. A total of 4278 proteins were identified, of which 1271 were differentially expressed. Additionally, 89 N-glycoproteins, 120 N-glycopeptides, and 128 N-glycosylation sites were detected. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed many MFGM proteins and N-glycoproteins involved in immune functions, mainly enriched in pathways such as complement and coagulation cascades, Fc gamma R-mediated phagocytosis, and hematopoietic cell lineage. Findings from this research offer unprecedented perspectives on the MFGM proteomes and their glycosylation in yak milk and support the development of functional dairy products.