Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Abstract

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of β- and α(S1)-casein during incubation of milk samples at peak somatic cell counts, with that of β-casein being faster than that of α(S1)-casein. Two-dimensional gel electrophoretograms of milk 6h after infusion with the toxin confirmed hydrolysis of β- and α(S1)-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C(18) columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from α(s1)- and β-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.

A peptidomic approach to biomarker discovery for bovine mastitis

“…There are only a very small number of studies in which peptide analysis has been performed on bovine milk samples during mastitis [15,25]. Both studies employed the same method, RP-HPLC, in separating the peptides, while further protein characterization was performed by MALDI-TOF/MS/ MS.…”

“…Following induction of mastitis by infusion of lipoteichoic acid (LTA), the toxin of S. aureus, Larsen et al [15] identified, by 2-D gel electrophoresis, hydrolysis of β-and α S1 -caseins 6 h after infusion with the toxin. Examination of the peptides at this and later time points, using reversedphase HPLC and MALDI-TOF MS/MS, detected increases in the milk peptidome with several different peptides being characterized and found to originate from α S1 -and β-caseins.…”

Comparison of the principal proteins in bovine, caprine, buffalo, equine and camel milk

“…2-DE was performed as described by Larsen et al (2010). Stained gels were then scanned using a calibrated GS-800 densitometer (Bio-Rad, Hercules, CA, USA)…”

Effects of duodenal infusion of free α-linolenic acid on the plasma and milk proteome of lactating dairy cows

“…Plasmin is primarily responsible for the observed proteolysis in milk with a low SCC, whereas cathepsins and elastase are implicated in proteolysis of milk with an elevated cell count (Wedholm et al, 2008). Increases in milk SCC (from 55 3 10 3 to 1382 3 10 3 cells/ml) have been correlated with increased plasmin, cathepsin D and cysteine protease activity (Somers et al, 2003), and with concomitant increases in proteolysis in milk, leading to the formation of a lower molecular weight casein fraction (Larsen et al, 2010). In our study, fragments of b-casein and a s1 -casein likely originated from increased caseinolysis in the milk of cows that received post-ruminal infusion of LNA at 400 g/day.…”