Multilevel Proteome Analysis Reveals the Region-specific Components of the Human Milk Fat Globule Membrane in China

Regional diversity may influence the composition of human milk fat globule membrane (HMFGM) protein composition and protein glycosylation. Here, we first systematically evaluated the efficiency and stability of six methods for the extraction of HMFGM proteins from breast milk. The optimal method (methanol-chloroform precipitation of MFGM proteins, followed by solubilization in 0.4% SDS lysis buffer) was further applied to the proteome and N-glycoproteome analysis of 49 breast milk samples from five regions across China. Proteomic analysis revealed the geographically divergent expression of BST2, COCH, FUCA1 and FGFBP1 in MFGM. The parallel N-glycoproteomic profiling identified 4914 site-specific N-glycans mapping to 689 glycoproteins, 699 glycosylation sites, and 420 glycan structures. This multiomics study reveals region-specific variations in the molecular composition of HMFGM proteins and their N-glycoprotein derivatives, elucidating the structural and functional dynamics of the human HMFGM in representative regions of China.