He Zhicong, Aristoteli Lina P, Kritharides Leonard, Garner Brett
School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
Biochem Biophys Res Commun. 2006 May 5;343(2):496-503. doi: 10.1016/j.bbrc.2006.03.007. Epub 2006 Mar 10.
Glycosylation is a common but variable modification that regulates glycoprotein structure and function. We combined small format 2D-PAGE with HPLC to analyse discrete human haptoglobin isoform N-glycans. Seven major and several minor haptoglobin isoforms were detected by 2D-PAGE. N-Glycans released from Coomassie-stained gel spots using PNGase were labeled at their reducing termini with 2-aminobenzamide. HPLC analysis of selected major isoform N-glycans indicated that sialic acid composition determined their separation by isoelectric focussing. N-Glycans from two doublets of quantitatively minor isoforms were also analysed. Although separation of each pair of doublets was influenced by sialylation, individual spots within each doublet contained identical N-glycans. Thus, heterogeneity in minor haptoglobin isoforms was due to modifications distinct from N-glycan structure. These studies describe a simple method for analysing low abundance protein N-glycans and provide details of discrete haptoglobin isoform N-glycan structures which will be useful in proteomic analysis of human plasma samples.
糖基化是一种常见但可变的修饰，可调节糖蛋白的结构和功能。我们将小型二维聚丙烯酰胺凝胶电泳（2D-PAGE）与高效液相色谱（HPLC）相结合，以分析离散的人触珠蛋白异构体N-聚糖。通过二维聚丙烯酰胺凝胶电泳检测到七种主要和几种次要的触珠蛋白异构体。使用肽-N-糖苷酶（PNGase）从考马斯亮蓝染色的凝胶斑点中释放的N-聚糖在其还原末端用2-氨基苯甲酰胺标记。对选定的主要异构体N-聚糖的高效液相色谱分析表明，唾液酸组成决定了它们通过等电聚焦的分离。还分析了来自两个定量次要异构体双峰的N-聚糖。虽然每对双峰的分离受唾液酸化影响，但每个双峰内的单个斑点含有相同的N-聚糖。因此，次要触珠蛋白异构体的异质性是由于不同于N-聚糖结构的修饰。这些研究描述了一种分析低丰度蛋白质N-聚糖的简单方法，并提供了离散触珠蛋白异构体N-聚糖结构的详细信息，这将有助于人血浆样品的蛋白质组分析。
