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

The proteic profiling of bovine milk produced by cows withsubclinical mastitis was obtained by MALDI mass spectrometry.A simple procedure of chemical fractionationof raw milk was developed, whereby less complex fractionsof proteins were obtained prior to mass spectrometricand SDS-PAGE analysis. The profiles of milk proteinsthus obtained could allow the identification of either earlymarkers of the acute phase of mastitis or endogenouspeptide of innate immune response. The activity of theendogenous proteases in raw milk produced from eachquarter of healthy and mastic cows was therefore assayedover 24-, 48-, 96-, and 216-h incubation at 37 °C at bothphysiological and acid pH. Sequence-specific peptideswere identified for each fraction by MS/MS experiments,and all tandem mass spectra were evaluated using MASCOTdatabase searching. The results show a specificproteolytic activity of endogenous enzyme toward β-caseinprecursor (P02666), α-S2-casein (P02663), α-S1-casein(P02662), and k-casein (P02668).

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-S-2-casein (P02663), alpha-S-1-casein (P02662), and kappa-casein (P02668).