Bovine milk proteome: Quantitative changes in normal milk exosomes, milk fat globule membranes and whey proteomes resulting from Staphylococcus aureus mastitis

Authors: Reinhardt, Timothy; Sacco, Randy; Nonnecke, Brian; Lippolis, John

Submitted to: Journal of Proteomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/9/2013
Publication Date: 2/13/2013
Publication URL: http://handle.nal.usda.gov/10113/61334
Citation: Reinhardt, T.A., Sacco, R.E., Nonnecke, B.J., Lippolis, J.D. 2013. Bovine milk proteome: Quantitative changes in normal milk exosomes, milk fat globule membranes and whey proteomes resulting from Staphylococcus aureus mastitis. Journal of Proteomics. 82:141-154.

Interpretive Summary: One of the greatest influences on milk composition, quality and therefore the milk’s consumer value is mastitis. Mastitis also results in the greatest economic loss to dairy farmers profitability. Comprehensive examination of the changes in the milk proteins that result from mastitis may increase our understanding of milk compositional effects on dairy food production, mammary biology, immune function in the mammary gland as well as identifying new biomarkers for early detection of this economically important disease effecting both the farmer and the dairy foods producer. This study examined the protein changes in bovine milk. The result was the identification of ~3000 proteins important for understanding milk and highlighted ~ 300 proteins that are significantly effected during mastitis. Many of these proteins are important to dairy product production and ~ 100 changed proteins have direct connections to the cows attempts to fight the infection, thus highlighting significant disease-fighting pathways that need to be further examined in real world mammary infections. These findings may result in a new and better understanding of the economically important dairy production disease mastitis.

Technical Abstract: Knowledge of milk protein composition/expression in healthy cows and cows with mastitis will provide information important for the dairy food industry, mammary biology and immune function in the mammary gland. To facilitate maximum protein discovery, milk was fractioned into whey, milk fat globule membranes (MFGM) and milk 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. Samples were subjected to offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to a tandem mass spectrometer. 2971 milk proteins combining the data from exosomes (2350 proteins), MFGM (1012 proteins) and whey (748 proteins) were identified with a false discovery rate of 0.1 %. 1490, 302 and 334 proteins were found to be unique to milk exosomes, MFGM and whey, respectively, under the conditions used. 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. This study provides new insights into mammary immune function and milk protein composition in changing conditions associated with infection.