Phosphorylation and glycosylation isoforms of bovine κ-casein variant E in homozygous Swedish Red cow milk by liquid chromatography-electrospray ionization mass spectrometry

Variations in the phosphorylation and glycosylation patterns of the common κ-casein (CN) variants A and B have been explored, whereas studies on variant E heterogeneity are scarce. This study reports for the first time the detailed phosphorylation and glycosylation pattern of the κ-CN variant E in comparison with variants A and B. Individual cow milk samples representing κ-CN genotype EE (n = 12) were obtained from Swedish Red cows, and the natural posttranslational modifications of its κ-CN were identified and quantified by liquid chromatography-electrospray mass spectrometry. In total, 12 unique isoform masses of κ-CN variant E were identified. In comparison, AA and BB milk consisted of 14 and 17 unique isoform masses, respectively. The most abundant κ-CN E isoform detected in the EE milk was the monophosphorylated, unglycosylated [1P 0G, ∼70%; where P indicates phosphorylation from single to triple phosphorylation (1–3P), and G indicates glycosylation from single to triple glycosylation (1–3G)] form, followed by diphosphorylated, unglycosylated (2P 0G, ∼12%) form, resembling known patterns from variants A and B. However, a clear distinction was the presence of the rare triphosphorylated, nonglycosylated (3P 0G, ∼0.05%) κ-CN isoform in the EE milk. All isoforms detected in variant E were phosphorylated, giving a phosphorylation degree of 100%. This is comparable with the phosphorylation degree of variants A and B, being also almost 100%, though with very small amounts of nonphosphorylated, glycosylated isoforms detected. The glycosylation degree of variant E was found to be around 17%, a bit higher than observed for variant B (around 14%), and higher than variant A (around 7%). Among glycosylation, the glycan e was the most common type identified for all 3 variants, followed by c/d (straight and branched chain trisaccharides, respectively), and b. In contrast to κ-CN variants A and B, no glycan of type a was found in variant E. Taken together, this study shows that the posttranslational modification pattern of variant E resembles that of known variants to a large extent, but with subtle differences.