Proteomics for milk proteins characterization in Girgentana goat breed

Abstract

Proteomics has been widely used to characterize milk protein content in livestock species and unravel protein biological functions. The aim of this work was to characterize the Girgentana goat breed whole milk and milk fraction proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. Bulk milk samples from two farms locate in Palermo and Agrigento (Sicily) areas were collected during the early stage of lactation. Initially, 2D-PAGE and protein detection protocols were set-up. In particular, 2D-PAGE analyses were carried out on whole milk using 18-cm IPG strips ranging from pH 3-10 and 4-7 and 12.5% polyacrylamide gel for first and the second dimension separation, respectively. Then, staining methods (silver-, colloidal Coomassie Blue G-250- and fluorescent dye-based method) were tested to reveal protein spots in 2D-gels. In silico 2D-gel image analysis, performed using Image Master 2D Platinum software, revealed that silver and fluorescent (based on SyproTM Ruby) staining methods showed similar results in terms of sensitivity/resolution. Then, centrifuge-based protocols were set-up to obtain milk protein fractions. The protein profiles of whole milk, caseins, whey and milk fat globule protein fractions were qualitatively compared in silico. This analysis showed that casein content decreased in both whey and fat globule protein fraction respect to caseins fraction which, instead, missed specific protein spots revealing in whey and/or fat globule fraction. Finally, a global protein differential expression analysis of whole milk samples from Palermo and Agrigento, respectively, was performed by 2D-Differential Gel Electrophoresis (2D-DIGE) using pH range 3-10. In silico analysis of the 2D-DIGE gel images (at least three per sample) showed a total of 143 differentially abundant spots with more than 1.5 fold change in volume and a statistically significant ANOVA value (p < 0.05) with 58 up-regulated and 84 down-regulated in Palermo farm milk respect to the Agrigento one. In addition, differential expression analysis will be also performed on milk fractions from Palermo and Agrigento, respectively, and the differentially abundant proteins and others showing invariant abundance will be identified by mass spectrometry to generate reference milk proteomic maps of Girgentana goat breed which could be useful to identify specific molecular marker assessing milk quality.