Analysis of O-glycosylation site occupancy in bovine κ-casein glycoforms separated by two-dimensional gel electrophoresis
Analysis of O-glycosylation site occupancy in bovine κ-casein glycoforms separated by two-dimensional gel electrophoresis
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DOI：
10.1002/pmic.200401098
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发表时间：
2005-03-01
期刊：
影响因子：
3.4
通讯作者：
Alewood, PF
中科院分区：
文献类型：
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作者：
Holland, JW;Deeth, HC;Alewood, PF
利用二维凝胶电泳（2-DE）分离糖蛋白的能力，分离出kappa-酪蛋白的不同糖型，这些糖型仅在附着的o聚糖数量上有所不同。为了确定聚糖的附着位置，用胃蛋白酶在凝胶中消化单个糖型，并在串联质谱（MS）中通过特征糖离子鉴定释放的糖肽。用氨/氨基乙硫醇代替甘聚糖后，用串联质谱法鉴定了o -糖基化位点。结果表明，在kappa-酪蛋白的5个潜在糖基化位点上，多糖并不是随机分布的。相反，单糖仿体的糖基化只能在单个位点T-152上检测到。类似地，二糖异构体似乎只在T-152和T-163位点被修饰，而三糖异构体在T-152、T-163和T-154位点被修饰。虽然不能排除其他位点的低糖基化水平，但糖型之间的位点占据层次结构是明显的，并证明了聚糖有序地添加到蛋白质中。由于所有五个潜在的o糖基化位点都可以在体内糖基化，因此某些位点在其他位点被占据之前似乎是潜伏的。通过2-DE分离的单个糖型中糖基化位点占用的测定揭示了一种不同的体内糖基化模式，这在以前没有被认识到。
The ability of two-dimensional gel electrophoresis (2-DE) to separate glycoproteins was exploited to separate distinct glycoforms of kappa-casein that differed only in the number of O-glycans that were attached. To determine where the glycans were attached, the individual glycoforms were digested in-gel with pepsin and the released glycopeptides were identified from characteristic sugar ions in the tandem mass spectrometry (MS) spectra. The O-glycosylation sites were identified by tandem MS after replacement of the glycans with ammonia/aminoethanethiol. The results showed that glycans were not randomly distributed among the five potential glycosylation sites in kappa-casein. Rather, glycosylation of the monoglycoform could only be detected at a single site, T-152. Similarly the diglycoform appeared to be modified exclusively at T-152 and T-163, while the triglycoform was modified at T-152, T-163 and T-154. While low levels of glycosylation at other sites cannot be excluded the hierarchy of site occupation between glycoforms was clearly evident and argues for an ordered addition of glycans to the protein. Since all five potential O-glycosylation sites can be glycosylated in vivo, it would appear that certain sites remain latent until other sites are occupied. The determination of glycosylation site occupancy in individual glycoforms separated by 2-DE revealed a distinct pattern of in vivo glycosylation that has not been recognized previously.