The relationships among bovine alpha(S)-casein phosphorylation isoforms suggest different phosphorylation pathways

Casein (CN) phosphorylation is an important post-translational modification and is one of the key factors responsible for constructing and stabilizing casein micelles. Variation in phosphorylation degree of aS-CN is of great interest because it is suggested to affect milk technological properties. This study aimed to investigate the variation in phosphorylation degree of alpha(S)-CN among milk of individual cows and to explore relationships among different phosphorylation isoforms of alpha(S)-CN. For this purpose, we analyzed morning milk samples from 529 French Montbeliarde cows using liquid chromatography coupled with electrospray ionization mass spectrometry. We detected 3 new phosphorylation isoforms: alpha(S2)-CN-9P, alpha(S2)-CN-14P, and alpha(S2)-CN-15P in bovine milk, in addition to the known isoforms alpha(S1)-CN-8P, alpha(S1)-CN-9P, alpha(S2)-CN-10P, alpha(S2)-CN-11P, alpha(S2)-CN-12P, and alpha(S2)-CN-13P. The relative concentrations of each alpha(S)-CN phosphorylation isoform varied considerably among individual cows. Furthermore, the phenotypic correlations and hierarchical clustering suggest at least 2 regulatory systems for phosphorylation of alpha(S)-CN: one responsible for isoforms with lower levels of phosphorylation (alpha(S1)-CN-8P, alpha(S2)-CN-10P, and alpha(S2)-CN-11P), and another responsible for isoforms with higher levels of phosphorylation (alpha(S1)-CN-9P, alpha(S2)-CN-12P, alpha(S2)-CN-13P, and alpha(S2)-CN-14P). Identifying all phosphorylation sites of alpha(S2)-CN and investigating the genetic background of different alpha(S2)-CN phosphorylation isoforms may provide further insight into the phosphorylation mechanism of caseins