Characterization of the Glycans of Equine κ‑Casein

To study the protein-bound glycans of equine κ-casein, equine sodium caseinate was first obtained from raw mare’s milk by acid precipitation and then fractionated by cation-exchange chromatography. The oligosaccharides of the obtained equine κ-casein were analyzed by RP-HPLC-UV-HRMS after β-elimination with simultaneous derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). In addition to the acidic tetrasaccharide derivative Neu5Ac-Gal-[Neu5Ac]-GalNAc-2PMP known from bovine κ-casein, the acidic pentasaccharide derivative Neu5Ac-Gal-[Gal-GlcNAc]-GalNAc-2PMP was identified as the most abundant glycan. The glycosylated amino acid residues were identified using a peptide sequencing approach after digestion with trypsin by HRMS. The threonine T109 was experimentally confirmed for the first time as a glycosylation site in equine κ-casein. Therefore, equine κ-casein seems to be more highly glycosylated than previously thought.