The correlation of milk peptide and protein profiling with the acute inflammatory stage in cows affected by mastitis

Correlation of protein and peptide profiling with the inflammatory stage for early diagnosis and treatment of mastitis. The quantity of the milk produced and its composition is altered in cows affected by mastitis. The inflammatory process associated to this disease, induces the chemical composition of milk approaches that of blood as a consequence of the increased permeability of blood mammary barrier. The intra-mammary infection process causes the elevation of SCC and increases proteolytic activity in milk. The correlation of protein and peptide profiling with the inflammatory stage can be a useful tool for a better understanding of the inflammatory processes, for early diagnosis and treatment of mastitis. The aim of this work is to endorse the changes in protein composition of milk during acute mastitis by applying a profiling of proteins and peptides by MALDI TOF/TOF. 50μl of milk was precipitated with 1ml of CHCl3/CH3OH 1:3 (v/v) affording to a liphophylic fraction (S) and pellet which was partitioned consecutively, under magnetic stirring and at r.t. with (1) 1ml of 50mM NH4HCO3, (2) 1ml di H2O, (3) 1ml of TFA (2%) and (4) 1.25ml di TFA (2%)/CH3CN 3:2 (v/v). 100μl of milk (a: pH =8; b: pH =6 ) was incubated for 24, 48 and 96h at 37 C, and than fractionated as reported above. The MALDI mass spectra were acquired on a 4700 Proteomics Analyzer mass spectrometer and MS/MS experiments were performed at collision energy of 1-2 kV. Protein identification was performed using MS and MS/MS search program from Matrix Science. The milk produced from all quarters (FL, FR, RL, RR) showing clinical symptom of acute mastitis were selected to determine the protein and peptide profile and to identify and characterize specific proteins without preventive chromatography or two dimensional gel separation. A simple procedure of chemical fractionation of whole milk was developed, whereby less complex fractions of proteolitic peptides and intact protein were obtained. All fractions were directly analyzed by MALDI TOF/TOF in the linear and reflectron mode. The protein content and assignment of each fraction was performed by MALDI MS in linear mode; fraction 1 contains ions specie at m/z 67148.56, 44724.39, 33446.38, 22276.36, 16704.67, 13348.14, 14181.26, 11631.85, 9139.97, 7087.95 and 6297.83 corresponding to [BSAH]+, [LactoferrinH]+, [BSAH]2+, [BSAH]3+, [BSAH]4+, [BSAH]5+, [alpha-LCAH]+, [CNH]2+, [beta-LGH]2+, [alpha-LCAH]2+ and to a unknown macro-peptide, respectively. Similar protein expression was observed in the other three fraction (2-4). Fraction 1 reveals the up-regulation of blood proteins and down regulation of serum protein induced by intra-mammary inflammation. Fraction S is the most interesting one, because it contains proteolytic peptides induced by the inflammatory processes; in fact a simple comparison between SFL, SFR, SRL and SRR, from the same cow, reflects the degree of the inflammations and let one to distinguish the most infected quarter. The incubation of milk at 37°C causes the formation of peptides in the range 1-5 KDa, whereas alpha-LCA and beta-LG are recovered intact even after 45h at 37°C. Although the peptide data set can be used for a statistical approach to early diagnosis of mastitis, the availability of a two-dimensional MS analysis provided by the TOF/TOF system, clearly indicates their protein precursors. Accordingly, k-CN, beta-CN, mithocondrial phosphate carrier protein precursor, alpha-S2 and alpha-S2A casein precursors, tropoelastine and serotransferrin have been easily recognized.