基于超滤膜辅助的糖蛋白全N-连接糖链的分离和质谱鉴定.docVIP

  基于超滤膜辅助的糖蛋白全 N-连接糖链的 分离和质谱鉴定# 杨刚龙，马恬然，李铮** 5 10 15 20 25 30 （西北大学生命科学学院，西安，710069） 摘要：【目的】建立一种基于超滤膜分离血清中糖蛋白全 N-连接糖链，并利用质谱技术对 糖链结构进行分析的方法。【方法】根据糖蛋白及其糖链结构之间的分子量差异，利用 Millipore 公司的 10KD 超滤膜分离血清糖蛋白上酶解（PNGase F）释放的全 N-连接糖链， 并使用 MALDI-TOF/TOF-MS 分析糖链结构。【结果】利用该技术可以从血清中分离并鉴定 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。【结论】该方法可 以被用于从大量生物样本中分离糖蛋白全 N-连接糖链，可以做到快速，高通量的分析糖蛋 白 N-连接糖链的目的。 关键词：生物化学和分子生物学；N-连接糖链；超滤膜辅助的糖蛋白全 N-连接糖链分离法； 超滤膜；糖蛋白；MALDI-TOF/TOF-MS 中图分类号：Q513 Isolation and Characterization of Total N-linked Glycans from Glycoproteins by Ultrafiltration Units and Mass Spectrometry YANG Ganglong, MA Tianran, LI Zheng (Life Science School, Northwest University,XiAn, 710069) Abstract: 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 isolation method (N-glycan-FASP-T) was developed using ultrafiltration units according to the molecular weight difference among the glycans, the impurities and proteins. Results: The isolated glycans were characterized and confirmed by the MALDI-TOF/TOF-MS. A total of 23 distinctive N-linked glycans were characterized from human sera. Key words: Biochemistry and molecular biology; N-linked glycans; Filter aided sample preparation-based total N-linked glycans from glycoproteins isolation method; Ultrafiltration unit; Glycoproteins; MALDI-TOF/TOF-MS 35 0 Introduction Glycosylation is one of the most common post-translational modifications in proteins. It has been estimated that over 50% of human proteins are glycosylated [1]. The glycans play important structural roles in protein folding, attaching proteins to cell membranes, protecting proteins from 40 degradation, and mediating many importa