Dephosphorylation of intact glycoprotein to greatly improve digestion efficiency coupled with matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometric analysis.
Dephosphorylation of intact glycoprotein to greatly improve digestion efficiency coupled with matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometric analysis.
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DOI：
10.1016/j.aca.2013.05.044
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发表时间：
2013-07
影响因子：
6.2
通讯作者：
Fenjie Li;Xiaodong Wang;Yujie Liu;Hui Liu;Zhili Li
中科院分区：
文献类型：
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作者：
Fenjie Li;Xiaodong Wang;Yujie Liu;Hui Liu;Zhili Li
唾液酸化对于多种细胞功能是必不可少的。在本论文中，我们以含有3个含复合型糖链结构的N-连接糖基化位点、4个O-连接糖基化位点和6个潜在磷酸化位点的牛胎蛋白为模型化合物，建立了一种高效的唾液酸糖蛋白消化策略和二氧化钛高效浓缩唾液酸化糖肽的策略。前者采用碱性磷酸酶消化、胰酶消化、蛋白酶K消化的工艺，可大大提高酶促胎球蛋白的效率，后者可明显提高以磷酸溶液为洗脱缓冲液的多唾液酸化糖肽的浓缩效率。从胎球蛋白中提取的浓缩糖肽的质谱图显示，几个离子簇的质量差为291Da，对应于多唾液酸化糖肽的存在。此外，应用该方法分别从健康人和性别、年龄匹配的甲状腺癌患者的血清中表征了α2-巨球蛋白和转铁蛋白的唾液酸化状态，它们的光谱表明，一个糖基化位点含有不同数量的唾液酸(SA)残基的糖形体的数量的变化可以用于区分健康人和癌症患者。
Sialylation is essential for a variety of cellular functions. Herein, we used bovine fetuin with three potential N-linked glycosylation sites containing complex-type glycan structures, four potential O-linked glycosylation sites and six potential phosphorylation sites as a model compound to develop a highly-efficient digestion strategy for sialylated glycoproteins and efficient enrichment strategy for sialylated glycopeptides using titanium dioxide. The former according to the process of alkaline phosphatase digestion followed by tryptic digestion and then proteinase K digestion could greatly improve the enzymatic efficiency on fetuin, and the latter could obviously enhance the enrichment efficiency for multisialylated glycopeptides using phosphoric acid solution as elution buffer. The mass spectra of the enriched glycopeptides derived from fetuin reveal that several series of the ion clusters with mass difference of 291Da correspond to the presence of multisialylated glycopeptides. In addition, the approach was applied to characterize the sialylated status of α2-macroglobulin and transferrin, respectively, from the sera of healthy subjects and sex- and age-matched patients with thyroid cancer, and their spectra indicate that the change in the amount of the glycoforms containing different number of sialic acid (SA) residues from one glycosylation site may be used to differentiate between healthy subjects and cancer cases.