title: Two-dimensional cartography of equine beta-casein variants achieved by isolation of phosphorylation isoforms and control of the deamidation phenomenon.
abstract: Because of variable degrees of phosphorylation, alternative splicing, and probable instability resulting from nonenzymatic deamidation, equine beta-casein presents a complex pattern by 2-dimensional electrophoresis that needs clarification. beta-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 beta-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 beta-casein pattern complexity. This phenomenon was strongly minimized when the first dimension step was carried out at 10 degrees C instead of at room temperature. Finally, spot attribution on 2-dimensional pattern of beta-casein was achieved by mixing each phosphorylation isoform in its native state with the whole beta-casein fraction.
authors.affiliation: Unité de Recherche Animal et Fonctionnalités des Produits Animaux - Equipe Protéolyse et Biofonctionnalités des Protéines et des Peptides, Nancy-Université, Vandoeuvre-lès-Nancy, France.
authors.forename: A
authors.initials: A
authors.lastname: Matéos
authors.forename: J-M
authors.initials: JM
authors.lastname: Girardet
authors.forename: D
authors.initials: D
authors.lastname: Mollé
authors.forename: A
authors.initials: A
authors.lastname: Dary
authors.forename: L
authors.initials: L
authors.lastname: Miclo
authors.forename: J-L
authors.initials: JL
authors.lastname: Gaillard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry*; D005591:Chemical Fractionation; D015180:Electrophoresis, Gel, Two-Dimensional; D005511:Food Handling; D006736:Horses; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020033:Protein Isoforms / Q000032:analysis; D013696:Temperature
chemical_list: D002364:Caseins; D020033:Protein Isoforms