Hykollari Alba, Malzl Daniel, Wilson Iain B H, Paschinger Katharina
Department für Chemie, Universität für Bodenkultur, Vienna, Austria.
Methods Mol Biol. 2019;1871:421-435. doi: 10.1007/978-1-4939-8814-3_24.
N-Glycans are posttranslational modifications of proteins attached to the amide side chains of asparagine residues, with possible heterogeneity due to different structures being possible at the same glycosylation site. In contrast to the mammalian systems, invertebrate N-glycosylation presents a challenge in analysis as there exist unfamiliar epitopes and a high degree of structural and isomeric variation between different species. A simple analytical approach to analyze N-glycans on specific glycoproteins is presented, which involves a combination of tryptic peptide mass spectrometry and "off-line" RP-HPLC MALDI-TOF MS/MS complemented by blotting to recognize specific epitopes. An additional N-glycan enrichment and labeling step can facilitate the analysis of single structures and even provide isomeric separation of N-glycans from specific proteins.
N-聚糖是连接到天冬酰胺残基酰胺侧链上的蛋白质的翻译后修饰，由于同一糖基化位点可能存在不同结构，因而可能具有异质性。与哺乳动物系统不同，无脊椎动物的N-糖基化在分析上具有挑战性，因为存在不熟悉的表位，并且不同物种之间存在高度的结构和异构体差异。本文介绍了一种分析特定糖蛋白上N-聚糖的简单分析方法，该方法涉及胰蛋白酶肽质谱分析与“离线”反相高效液相色谱-基质辅助激光解吸电离飞行时间串联质谱（RP-HPLC MALDI-TOF MS/MS）相结合，并通过印迹法识别特定表位。额外的N-聚糖富集和标记步骤可以促进单一结构的分析，甚至可以实现特定蛋白质N-聚糖的异构体分离。
