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. 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. Published by Elsevier B.V.