基于超滤膜辅助的糖蛋白全N-连接糖链的富集和质谱解析

糖基化作为一种常见的蛋白质翻译后修饰,对蛋白质的空间结构、生物功能等具有重要的影响.解析糖蛋白糖链结构有助于更清楚地认识糖蛋白及其功能.本研究建立了一种基于超滤膜富集血清中糖蛋白全N-连接糖链,并利用质谱技术对糖链结构进行分析的方法.根据糖蛋白及其糖链结构之间的分子质量差异,利用Millipore公司的10 ku超滤膜富集血清糖蛋白上酶解(PNGase F)释放的全N-连接糖链,并使用MALDI-TOF/TOF-MS解析糖链结构.通过该技术可以从血清中富集并鉴定到23种独特的N-连接的糖链结构,并且利用二级质谱进行了结构确认.该方法可以被用于从大量生物样本中富集糖蛋白全N-连接糖链,可以达到快速、高通量地解析糖蛋白N-连接糖链的目的.

Enrichment and Characterization of Total N-linked Glycans From Glycoproteins by Ultrafiltration Units and Mass Spectrometry

Glycosylation is one of the most common post-translational modifications in proteins. Current methods for glycan analysis are generally based on multiple preparation processes to separate glycans. However, glycans are continuously lost and the difficulty for accurate quantitative analysis is increased in the procedure. Here, a filter aided sample preparation-based total N-linked glycans from the glycoproteins enrichment and separation method (N-glycan-FASP-T) was developed using ultrafiltration units according to the molecular mass difference among the glycans, the impurities and proteins. The enriched glycans were characterized and confirmed by the MALDI-TOF/TOF-MS. A total of 23 distinctive N-linked glycans were characterized from human serum.