Proteomic characterization of donkey milk “caseome”

At present, compared with bovine milk, the characterization of donkey milk caseins is at a relatively early stage progress, and only limited data are related to its genetic polymorphism. In this work, the heterogeneity of donkey caseome was investigated using a proteomic approach, based on one- (PAGE, UTLIEF) and two-dimensional (PAGE → UTLIEF) electrophoresis, stained with either Coomassie Brilliant Blue or specific polyclonal antibodies, and structural MS analysis. These combined methodologies allowed the contemporary identification of donkey α_s1, α_s2, β and κ-CN with their related heterogeneity due to phosphorylation (α_s1, α_s2 and β-CN), glycosylation (κ-CN) and incorrect splicing of RNA in mRNA (deleted forms of α_s1-CN and β-CN). The results achieved showed 11 components for κ-CN, six phosphorylated components for β and α_s1-CN and three main phosphorylated components for α_s2-CN, each accounting for 10, 11 and 12 P/mole. At this regard, for the first time, the primary structure of the expressed protein corresponding to the only available donkey α_s2-CN cDNA sequence was determined. Furthermore β-CN was found in homozygous and heterozygous state for the occurrence of a genetic β-CN variant having a MW value 28 mass units higher than the common β-CN phenotype.