Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, China.
Analyst. 2011 Nov 7;136(21):4422-30. doi: 10.1039/c1an15554g. Epub 2011 Sep 7.
Characterization of protein glycosylation requires highly specific methods for the enrichment of glycopeptides because of their sub-stoichiometric glycosylation-site occupancy. The hydrophilic affinity based strategy has attracted more attention, owing to its broad glycan specificity, good reproducibility, and compatibility with mass spectrometric (MS) analysis. Several polar matrices have emerged for hydrophilic interaction chromatography (HILIC) approaches, including sepharose, cellulose, ZIC-HILIC and titania. Here, we present the solid-phase extraction (SPE) utility of zirconia coated mesoporous silica (ZrO(2)/MPS) microspheres for glycopeptide isolation prior to MS analysis. The high specificity of this SPE approach was demonstrated by the enrichment of glycopeptides from the digests of model glycoproteins in HILIC mode. ZrO(2)/MPS microspheres show superior selectivity and glycosylation heterogeneity coverage for glycopeptide enrichment to conventional sepharose. Furthermore, digested mixtures of the phosphoprotein α-casein and IgG were also treated with ZrO(2)/MPS HILIC SPE materials, which exhibited that glycopeptides could be effectively enriched with interference from phosphorylated peptides.
蛋白质糖基化的特征需要高度特异性的方法来富集糖肽，因为它们的糖基化位点占有率是亚化学计量的。由于其广泛的聚糖特异性、良好的重现性和与质谱 (MS) 分析的兼容性，基于亲水性亲和力的策略引起了更多的关注。已经出现了几种用于亲水相互作用色谱 (HILIC) 方法的极性基质，包括琼脂糖、纤维素、ZIC-HILIC 和二氧化钛。在这里，我们介绍了涂有氧化锆的介孔硅 (ZrO(2)/MPS) 微球在 MS 分析之前用于糖肽分离的固相萃取 (SPE) 用途。这种 SPE 方法的高特异性通过在 HILIC 模式下从模型糖蛋白的消化物中富集糖肽得到了证明。ZrO(2)/MPS 微球在糖肽富集方面表现出优于传统琼脂糖的选择性和糖基化异质性覆盖。此外，还对磷酸化蛋白 α-酪蛋白和 IgG 的消化混合物进行了 ZrO(2)/MPS HILIC SPE 处理，结果表明可以有效地从磷酸化肽的干扰中富集糖肽。
