Laboratory of Glyco-Organic Chemistry, The Noguchi Institute, 1-9-7 Kaga, Itabashi-ku, Tokyo, Japan.
Laboratory of Glyco-Organic Chemistry, The Noguchi Institute, 1-9-7 Kaga, Itabashi-ku, Tokyo, Japan.
Carbohydr Res. 2020 May;491:107981. doi: 10.1016/j.carres.2020.107981. Epub 2020 Mar 19.
O-Glycopeptides derived from natural bioresources are an attractive material for a variety of purposes. Whey protein products are used as a human dietary supplement and in animal feed and are a readily available resource for the preparation of O-glycopeptides. The protein composition of bovine milk is well-studied, and many glycoproteins carrying N-glycans and O-glycans have been found in commercial whey protein products. In particular, κ-casein glycomacropeptide and lactophorin, which have several O-glycans, are known to exist in whey protein. Here, we report an isolation method of O-glycopeptides bearing disialyl core 1 type and core 2 type glycan moieties from commercially available whey protein products using proteose peptone extraction, enzymatic digestion (with trypsin or thermolysin), and sequential high-performance liquid chromatography purification. We were able to isolate several kinds of O-glycopeptides from lactophorin and κ-casein: six peptide sequences and five kinds of O-glycans. The O-glycopeptides were detected and identified by flow injection analysis combined with electrospray ionization mass spectrometry and tandem mass spectrometry using collision-induced dissociation and electron transfer dissociation. O-Glycopeptides bearing a variety of O-glycans could be used as a substrate for endo-α-N-acetyl galactosaminidase, and their various O-glycan structures were useful for the investigation of enzyme activities.
来源于天然生物资源的 O-糖肽是各种用途的有吸引力的材料。乳清蛋白产品用作人类膳食补充剂和动物饲料，并且是制备 O-糖肽的易得资源。牛乳的蛋白质组成得到了很好的研究，并且在商业乳清蛋白产品中发现了许多携带 N-聚糖和 O-聚糖的糖蛋白。特别是，具有几个 O-聚糖的κ-酪蛋白糖巨肽和乳白蛋白已知存在于乳清蛋白中。在这里，我们报告了一种使用蛋白酶解（胰蛋白酶或糜蛋白酶）和顺序高效液相色谱纯化从市售乳清蛋白产品中分离带有二唾液酸核心 1 型和核心 2 型聚糖部分的 O-糖肽的方法。我们能够从乳白蛋白和κ-酪蛋白中分离出几种 O-糖肽：六种肽序列和五种 O-聚糖。通过使用流注射分析结合电喷雾电离质谱和串联质谱（使用碰撞诱导解离和电子转移解离），检测和鉴定了 O-糖肽。具有各种 O-聚糖的 O-糖肽可以用作内切-α-N-乙酰半乳糖胺酶的底物，并且它们的各种 O-聚糖结构对于研究酶活性很有用。
