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Title: Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced Escherichia coli mastitis

Author
item BOEHMER, JAMIE - US FOOD & DRUG ADMIN
item Bannerman, Douglas
item SHEFCHECK, KEVIN - US FOOD & DRUG ADMIN
item WARD, JEFFREY - US FOOD & DRUG ADMIN

Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/25/2008
Publication Date: 11/20/2008
Citation: Boehmer, J.L., Bannerman, D.D., Shefcheck, K.J., Ward, J.L. 2008. Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced Escherichia coli mastitis. Journal of Dairy Science. 91(11):4206-4218.

Interpretive Summary: Mastitis remains a prevalent disease in dairy cows and is one of the most costly diseases to the animal agricultural industry. Escherichia coli are among the most prevalent bacteria to induce clinical mastitis. The objective of the current study was to evaluate a method requiring minimal sample preparation so as to enable rapid and cost effective identification of potential biomarkers of mastitis. The major finding of this study was that the use of 2-dimensional gel electrophoresis (2D-GE) coupled with peptide sequencing using matrix-assisted laser desorption/ ionization-time of flight (MALDI-TOF) mass spectrometry post source decay (PSD) is an effective method for identifying changes in milk protein expression during mastitis. The results of this study are important to scientists, the dairy industry, and veterinarians because they establish a method for identifying milk biomarkers of mastitis that may be used to evaluate the efficacy of drugs developed to treat this disease. Further, this method has the potential to identify differentially expressed proteins that may confer susceptibility and/or resistance to mastitis.

Technical Abstract: The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis (2D-GE) on whey samples from a group of 8 cows prior to and 18 hours after infection with Escherichia coli, and to identify differentially expressed milk proteins by peptide sequencing using matrix-assisted laser desorption/ ionization-time of flight (MALDI-TOF) mass spectrometry post source decay (PSD). Only proteins present in whey fractions of all 8 cows were sequenced to avoid reporting a protein response unique to only a subset of infected cows. Despite the overwhelming presence of casein and beta-lactoglobulin, the low abundance proteins transthyretin, lactadherin, beta-2-microglobulin precursor, and complement C3 precursor could be identified in whey samples from healthy cows. Whey samples at 18 hours post infection were characterized by an abundance of serum albumin, in spots of varying mass and isoelectric point, as well as increased transthyretin and complement C3 precursor levels. Also detected at 18 hours post inoculation were the antimicrobial peptides cathelicidin, indolicidin, and bactenicin 5 and 7, and the proteins ß-fibrinogen, a-2-HS-glycoprotein, S100-A12, and a-1-antiproteinase. Most notable was the detection of the acute phase protein a-1 acid glycoprotein in both healthy and mastitic milk samples. In contrast to methods used in previous proteomic analyses of bovine milk, the methods used in the current study enabled the rapid identification of milk proteins with minimal sample preparation. Use of a larger sample size than previous analyses also allowed for more robust protein identification. Results indicate that examination of the protein profile of whey samples from cows following inoculation with E. coli could provide a rapid and comprehensive survey of milk protein modulation during coliform mastitis and aid in the identification of biomarkers of this disease.