Exploitation of endogenous protease activity in raw mastitic milk by MALDI-TOF/TOF

The proteic profiling of bovine milk produced by cows with subclinical mastitis was obtained by MALDI mass spectrometry. A simple procedure of chemical fractionation of raw milk was developed, whereby less complex fractions of proteins were obtained prior to mass spectrometric and SDS-PAGE analysis. The profiles of milk proteins thus obtained could allow the identification of either early markers of the acute phase of mastitis or endogenous peptide of innate immune response. The activity of the endogenous proteases in raw milk produced from each quarter of healthy and mastic cows was therefore assayed over 24-, 48-, 96-, and 216-h incubation at 37 degrees C at both physiological and acid pH. Sequence-specific peptides were identified for each fraction by MS/MS experiments, and all tandem mass spectra were evaluated using MASCOT database searching. The results show a specific proteolytic activity of endogenous enzyme toward beta-casein precursor (P02666), alpha-S2-casein (P02663), alpha-S1-casein (P02662), and kappa-casein (P02668).