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

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.