Characterisation of the heterogeneity of ovine deleted variant alphaS1-casein E by a proteomic approach

The micro-heterogeneity of the ovine αs1-CN E variant, occurring in Leccese ewe milk, was characterized using a bottom-up, shotgun proteomic approach by means of two-dimensional gel electrophoresis (2-DE) and RP-HPLC as separation techniques and polyclonal antibodies against αs1-CN and nano-liquid chromatography-electrospray ionization-time-of-flight tandem mass spectrometry nESI-QTOF/MS/MS as an identification methodology. Comparing results with the most common genetic variant αs1-CN C, αs1-CN E variant showed a reduced number of phosphorylated serine residues (αs1-CN E -4P vs αs1-CN C -9P) and a lower measured molecular weight (MW) (22241.7Da vs 23481.5Da). The MS analysis of protein tryptic digest ascertained that these differences were due to the αs1-CN (70-77) amino acid sequence deletion, coded by exon 10, thus triggering the phosphate loss on serine residues located at positions 64, 66, 68 and 75. The loss of four phosphate groups, associated with the lower content of αs1-CN E, could be negatively related both to cheese-making aptitude of milk and mineral carrier activity.