Bovine mastitis is caused by a wide range of pathogens which results in a substantial economic loss for the dairy cattle livestock industry. β-defensins are a part of the innate immune system and act as the first line of defense against mastitis in bovine. β-defensins (~6-10kDa) are antimicrobial peptides that contain about 28 to 67 amino acids, the presence of six cysteine residues results in three disulfide bonds formed and a β-sheet structure. A few β-defensins; TAP (Tracheal antimicrobial peptide), LAP (Lingual antimicrobial peptide), DEFB1 (β defensin 1 peptide), DEFB3 (β defensin 3 peptide), DEFB4 (β defensin 4 peptide), DEFB5 (β defensin 5 peptide), DEFB10 (β defensin 10 peptide), and DEFB103 (β defensin 103 peptide) have been identified in cows based on site of occurrence and time of expression in tissue, but not in milk. A Solid Phase Extraction (SPE) and Liquid Chromatography Mass Spectrometry (LCMS) method is developed for the identification of β-defensin in bovine milk of healthy and mastitis-infected cows. The LC-MS response for infected bovine milk was compared with the response obtained from the healthy sample. The peptides were separated on a Phenomenex bioZen Intact XB-C8 column (150 x 2.1 mm, 3.6 µm) on a Thermo Scientific HPLC system using an acetonitrile:H<sub>2</sub>O in 0.1% formic acid mobile phase gradient coupled with the TSQ Thermo Fortis mass spectrometer. A scan range from 100 –2000 m/z in the positive ion mode is used for the acquisition. Other antimicrobial β-defensin peptides are expected in mastitis-infected bovine milk and will be explored.

Liquid chromatography mass spectrometry method development for determination of β-defensins in bovine milk

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