RESUMO
The efficiency of reversed-phase HPLC, capillary electrophoresis (CE), PAGE and isoelectric focusing with immunoblotting in separating ovine caseins has been evaluated. The assessment was carried out by employing electrospray ionization-mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionization-time of flight as reference tools for identifying protein components. Ovine casein was fractionated by HTPC into four major peaks. With ESI-MS, each peak contained components belonging to only one of the four casein families. On-line liquid chromatography-ESI-MS allowed us to determine each fraction's composition by detecting thirteen alphas1-, eleven alphas2-, seven beta-, and three kappa-casein (CN) components. The alphas1-CN and alphas2-CN consisted of eight and two protein chains respectively of lengths differing through the deletion of one or more peptide sequences; they were also discretely phosphorylated as kappa-CN and beta-CN. By CE at pH 2.5, each casein fraction was as heterogeneous as that resulting from ESI-MS for the single HPLC-derived fractions. The separation of alphas1-CN and alphas2-CN proved to be excellent, with the exception of a co-migration of kappa0-CN with a minor alphas1-CN component and of a glycosylated kappa-CN for with low-phosphorylated = alphas1-CN and beta-CN components. Dephosphorylation of whole casein was used to reduce the heterogeneity of the native fractions and by applying currently used analytical techniques it was possible to visualize the protein moiety difference along the CE profile. CE, HPLC, and immunoblotting were all equally capable of effecting an accurate separation of the four dephosphorylated casein families. The spectra obtained by ESI-MS directly on dephosphorylated whole ovine casein samples contained the signals of the four casein families and the relative alphas1-CN variants, the non-allelic alphas1-CN and alphas2-CN forms, dimeric kappa-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.
