Proteomic investigation of differentially expressed proteins in buffalo (Bubalus bubalis) milk whey during mastitis

The aim of this study was to investigate, by performing conventional 2D-electrophoresis (2-DE) and 2D-DiGE, modifications of the milk whey proteome profile in buffaloes during mastitis. Milk samples were collected from healthy (G1, negative bacteriology, SCC 100,000 cells/mL) buffaloes. To perform 2-DE, samples with low (0.10-0.50 μg/mL, n=6) and high (1.41-12.08 μg/mL, n=12) haptoglobin levels were selected from G1 and G2, respectively. To perform 2D-DiGE, pools with low (0.10-0.44 μg/mL) and high haptoglobin levels (5.98-12.08 μg/mL) were prepared using milk whey samples from G1 and G2, respectively. 2-DE was accomplished by loading 200μg of total protein of each sample into 11cm, pH3-10 IPG strips, followed by SDS-PAGE. 2D-DiGE was accomplished by loading 50μg of total protein of two different pools (healthy vs mastitis), labelled with fluorescent dyes (Cy3, Cy5), into 24cm pH4-7 IPG strips, followed by SDS-PAGE. Gel images were analyzed using SameSpot (2-DE) and DeCyder software (2D-DiGE). Spots of interest were excised and subjected to tryptic in-gel digestion and analysed by LC-ESI-MS/MS. Protein identifications were assigned using NCBI databases. 2-DE highlighted ten spots differently expressed during mastitis. From these, seven spots were increased, where host-defence proteins (lactoferrin, complement C3, imunoglobulin light chain) and clusterin were identified. Three spots were decreased, where high abundance proteins (β-Lactoglobulin, α-Lactalbumin) were identified. 2D-DiGE highlighted 29 spots differently expressed during mastitis. From these, 15 spots were increased, where host-defense proteins (lactoferrin, complement C9, imunoglobulin light chain, endopin 2B and apolipoprotein A1) and clusterin were identified. 14 spots, identified as β-lactoglobulin and α-lactalbumin were decreased. In conclusion, 2-DE and 2D-DiGE allowed a comparison of protein spots between healthy and mastitic buffalo milk whey samples and the two techniques combined could give important information regarding potential biomarkers of mastitis. This project received financial support from FAPESP (2013/26498-5) and University of Glasgow.