Identification of ovine casein heterogeneity by HPLC and electrospray mass spectrometry

The structural features of the single protein species occurring in each casein family have been determined using an analytical procedure consisting of HPLC in the first dimension and electrospray mass spectrometry in the second. Native casein was isolated by HPLC on a C18 column and submitted to ES/MS analysis. In order to determine the number of phosphate groups, each HPLC peak was submitted to dephosphorylation and molecular mass determined. On the basis of the molecular mass shift and knowledge of the amino acid sequence, the casein composition was obtained. A sample of individual ovine casein was found to be composed of (i) four alphas1-Cn A forms of which one was 199-residue long at four phosphorylation levels and three shorter non-allelic forms at different level of phosphorylation; (ii) two non-allelic forms of alphas1-Cn at five and six phosphorylation levels; (iii) six multiphosphorylated beta-Cn forms; and (iv) three multiphosphorylated kappa-Cn forms.