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Two-dimensional cartography of equine β-casein variants achieved by isolation of phosphorylation isoforms and control of the deamidation phenomenon

A. Matéos^*, J.-M. Girardet^*,1, D. Mollé, A. Dary^*, L. Miclo^* and J.-L. Gaillard^*

^* Unité de Recherche Animal et Fonctionnalités des Produits Animaux (UR AFPA) – Equipe Protéolyse et Biofonctionnalités des Protéines et des Peptides (PB2P), Nancy-Université, Vandœuvre-lès-Nancy, France
Science et Technologie du Lait et de l’uf (STLO), INRA UMR 1253, Rennes, France

¹ Corresponding author: jean-michel.girardet{at}scbiol.uhp-nancy.fr

Because of variable degrees of phosphorylation, alternative splicing, and probable instability resulting from nonenzymatic deamidation, equine β-casein presents a complex pattern by 2-dimensional electrophoresis that needs clarification. β-Casein prepared from Haflinger mare’s milk by hydrophobic interaction chromatography was fractionated by ion-exchange chromatography according to the degree of phosphorylation. Isoforms were identified by mass spectrometry; they corresponded to the full-length protein having 3 to 7 phosphate groups and to the splicing variant involving exon 5 and containing 4 to 7 phosphate groups. Investigations of nonenzymatic deamidation showed that β-casein did not deamidate spontaneously in stored milk and during the different steps of chromatography, but deamidation could occur when 2-dimensional electrophoresis was performed, increasing the β-casein pattern complexity. This phenomenon was strongly minimized when the first dimension step was carried out at 10°C instead of at room temperature. Finally, spot attribution on 2-dimensional pattern of β-casein was achieved by mixing each phosphorylation isoform in its native state with the whole β-casein fraction.

Key Words: equine β-casein • deamidation • phosphorylation • two-dimensional cartography