基于固相萃取的微量糖蛋白糖基化修饰表征技术的建立

Characterization of N-Glycosylation Status of Micro-level Glycoprotein Using Solid Phase Extraction Based Techniques

结合自制亲水固相萃取富集柱和生物质谱鉴定技术,实现了糖基化蛋白质核糖核酸酶B的糖含量测定、糖基化位点确认、聚糖富集及结构表征,以及不同糖型相对丰度分析。结果表明:其糖含量8.47%,糖基化位点为34位的Asn,糖链主要为5种高甘露糖型结构(Man5-9GlcNAc2)。所建立的HILIC富集技术,有利于针对微量生物样本,如生物工程药物糖蛋白及重要功能糖蛋白,开展位点特异性糖链结构解析,为糖蛋白质的药效或功能研究提供线索。

A new strategy integrating the latest hydrophilic interaction chromatographic （HILIC） method with mass spectrometry technologies was developed for the enrichment and determination of intact glycopeptides and glycan structures from a glycoprotein. The average molecular weight, the ratio of glycosylation, the glycosite and the structures of glycoforms of ribonuclease B （RNaseB） were determined by MALDI-TOF-MS and ESI-LTQ-FT-MS. RNaseB contains 8. 43% glycan and has a single glycosylation site at Asn34 with the glycan structure varied from five to nine mannose residues （Man5-9GlcNAc2）. The strategy paves the way for systematic and confident analysis of complex glycoproteins with important biological or pharmaceutical functions.