Deep 4D DIA-PASEF proteomics profiling uncovers processing-dependent differences between milk fat globule membrane and extracellular vesicles

Milk fat globule membrane (MFGM) and extracellular vesicles (EVs) are key bioactive dairy components critical for advanced functional foods and next-generation infant formula, yet their molecular compartmentalisation across industrial processing streams remains poorly defined. Using a high-resolution 4D DIA-PASEF workflow, we profiled three industrial fractions (acid whey powder, buttermilk powder, and whey-fat concentrate (WFC)) against laboratory-isolated benchmarks (MFGM-lab and EV-lab). Achieving an extensive depth of 3792 protein groups across all samples, this unbiased mass spectrometry approach successfully mitigated high-abundance masking, capturing low-abundance receptors and vesicle trafficking proteins (e.g., VAMP2, IGF2R). The data revealed distinct processing-dependent proteomic shifts. Mild, solvent-free laboratory isolation effectively retained the intracellular biosynthetic and transport apparatus associated with the endoplasmic reticulum and lipid droplets. In contrast, industrial manufacturing altered the spatial distribution of these proteins based on specific processing lines. Crucially, aligned with MISEV2018 guidelines and BTN1A1/CD9 abundance ratios, we demonstrate that physical phase partitioning during casein coagulation and removal concentrates a high relative proportion of EV-associated proteins into commercial WFC. Rather than presenting an exclusive loss of membrane structures, WFC exhibits a unique hybrid molecular profile that inherently co-enriches both vesicular and MFGM proteins. Ultimately, these molecular-level insights provide an empirical foundation for a strategic shift in dairy processing, guiding the targeted valorisation of commercial whey fractions from generic bulk commodities into precision-targeted functional dairy ingredients.