Milk protein expression in healthy cows and cows with
mastitis will provide information important for the dairy food industry and immune function in the mammary gland. To facilitate
protein discovery, milk was fractioned into whey,
milk fat globule membranes (MFGM) and exosomes from healthy and Staphylococcus aureus infected cows.
Amine-reactive isobaric tags (iTRAQ) were used to quantify
protein changes between milk fractions isolated from healthy and S. aureus infected cows. 2971
milk proteins were identified with a false discovery rate of 0.1%. Greater than 300
milk proteins associated with host defense were identified and 94 were significantly differentially regulated in S. aureus infected milk compared to their uninfected controls. These differentially regulated host defense
proteins were selectively segregated in the 3 milk compartments examined. An example of this segregation of host defense
proteins was the partitioning and high concentration of
proteins indicative of neutrophil extracellular traps (NETs) formation in the MFGM preparations from S. aureus infected milk as compared to exosomes or whey.
Protein composition changes found in milk exosomes, MFGM and whey during an
infection provides new and comprehensive information on
milk protein composition in general as well as changes occurring during an
infection.
BIOLOGICAL SIGNIFICANCE: The significance of this study is the identification and quantification of the individual components of the neutrophil extracellular traps (NET) functional
proteome in an apparent stable complex with MFGM and/or milk fat globules during an intra-mammary
infection. NETs could be functionally relevant in intra-mammary
infection, as it is known that during an
infection neutrophils ingest large amounts of milk fat that down regulates many of their traditional immune functions. Thus the presence of NETs in milk fat provides new insights to mammary immune function and suggests a role for NETs in clinical
mastitis. These in vivo NETs can now be tested to determine if they retain functional antimicrobial activity when primarily associated with milk fat. Then we can estimate their real world functional relevance during an intra-mammary
infection, which is one key to understanding clinical
mastitis in dairy cows.