Equine αS1-casein: Characterization of alternative splicing isoforms and determination of phosphorylation levels

Abstract

αS1-Casein was isolated from Haflinger mare's milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare's αS1-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete de-phosphorylation. The apoforms were then analyzed by electrospray ionization mass spectrometry. The results revealed the existence of a full-length protein and 7 variants resulting from posttranscriptional modifications; that is, exon skipping involving exon 7, exon 14, or both and use of a cryptic splice site encoding a glutamine residue. The determination of the different phosphorylation degrees of the native isoforms of αS1-casein was finally achieved by electrospray ionization mass spectrometry analysis after fractionation of the isoforms by ion-exchange chromatography. Thus, 36 different variants of equine αS1-casein were identified with several phosphate groups ranging from 2 to 6 or 8 depending on whether exon 7 was skipped. © American Dairy Science Association, 2009.