Chair of Food Chemistry, Technische Universität Dresden, 01062 Dresden, Germany.
J Agric Food Chem. 2023 Jun 21;71(24):9460-9468. doi: 10.1021/acs.jafc.3c00452. Epub 2023 Jun 6.
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.
为了研究马κ-酪蛋白的糖基化蛋白质，首先通过酸沉淀从马乳中获得马酸钠酪蛋白，然后通过阳离子交换色谱进行分级。用 1-苯基-3-甲基-5-吡唑啉酮（PMP）同时衍生化进行β消除后，用 RP-HPLC-UV-HRMS 分析获得的马κ-酪蛋白的寡糖。除了从牛κ-酪蛋白中已知的酸性四糖衍生物 Neu5Ac-Gal-[Neu5Ac]-GalNAc-2PMP 外，还鉴定出酸性五糖衍生物 Neu5Ac-Gal-[Gal-GlcNAc]-GalNAc-2PMP 作为最丰富的聚糖。用胰蛋白酶消化后，通过 HRMS 进行肽测序，确定糖基化氨基酸残基。实验首次证实马κ-酪蛋白中的苏氨酸 T109 是糖基化位点。因此，马κ-酪蛋白似乎比以前认为的更高度糖基化。
