Two-dimensional difference in gel electrophoresis (2D-DIGE) for milk proteins characherization in Girgentana goat breed

Abstract

Milk proteomics has rapidly become an eligible approach, since the protein fraction constitutes the most biologically relevant component in milk. Proteomic applications can vary greatly from straightforward protein identification to complex characterization of post-translational modifications and protein-protein interactions. The aim of this work was to compare protein profile of Girgentana whole milk samples between lactation periods and geographical areas by two dimensional difference in gel electrophoresis (2D-DIGE). Milk samples were collected in two flocks located in two different areas of Sicily at three different time points from calving: 30, 55, and 120 days, respectively. Protein pools were labelled with CyDye fluorophores Cy3, Cy5, and the internal standard was labelled with Cy2. Electrophoresis was carried out using pH range 3-10 in the first dimension and 12.5% polyacrylamide gel to separate proteins in the second dimension. Image and statistical analyses were performed using Image Master 2D Platinum software. Several spots on the gels appeared as strings, demonstrating that isoforms of differing charge, resulting from post-translational modifications, are present in milk. Protein spots showing more than 1.5 fold change in spot volume (increased for up-regulation or decreased for down-regulation), with a statistically significant ANOVA value (P≤0.05), were considered differentially abundant. Among these, a set of ten spots were excised from preparative gels and identified by liquid chromatography tandem mass spectrometry. To assign protein identity, data were filtered following the application of identification criteria based on number of unique peptides ≥2. Milk fat globule EGF factor 8 protein variants, β-lactoglobulin, β-casein and serum albumin were successfully identified. 2D-DIGE allowed giving a general picture of milk protein distributions over 3-10 pH range. These preliminary results in addition to the identification of others interesting spots will be used to generate a reference proteomics map for the Girgentana goat breed.