Mass spectrometry-based procedure for the identification of ovine casein heterogeneity

Abstract

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 HPLC 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 α_s1-, eleven α_s2-, seven β-, and three κ-casein (CN) components. The α_s1-CN and α_s2-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 κ-CN and β-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 α_s1-CN and α_s2-CN proved to be excellent, with the exception of a co-migration of κ₀-CN with a minor α_s1-CN component and of a glycosylated κ-CN form with low-phosphorylated α_s1-CN and β-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 α_s1-CN variants, the non-allelic α_s1-CN and α_s2-CN forms, dimeric κ-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.