基于固定化Pronase E酶与质谱的糖链结构分析方法  

Combination of immobilized Pronase E digestion and mass spectrometry for the analysis of glycoforms

作　　者：张曼丽[1,2] 于子翔[1,2] 钱小红[2] 应万涛[2] 

机构地区：[1]安徽医科大学军事医学科学院,合肥230032 [2]蛋白质组学国家重点实验室,北京蛋白质组研究中心,军事医学科学院放射与辐射医学研究所,北京102206

出　　处：《分析试验室》2016年第1期1-6,共6页Chinese Journal of Analysis Laboratory

基　　金：国家高科技研究发展计划(2012AA020203);北京市科技新星计划(Z121107002512014)资助

摘　　要：位点特异性糖链结构的解析,是糖蛋白质结构分析面临的巨大挑战。Pronase E蛋白水解酶,能够降解糖蛋白或糖肽中大部分的氨基酸序列,而保留糖链与少量氨基酸的序列,与色谱、质谱分析等联用,可以实现糖链结构的鉴定,同时,保留的氨基酸序列可以辅助实现修饰位点的识别,两者结合,可以获取位点特异性糖链结构的信息。但Pronase E酶解的缺点是,酶解效率较低,常需要较高浓度的蛋白酶。本实验将Pronase E固定化在基质上,固定化后的Pronase E具备较高的局部浓度,从而实现目标糖蛋白的快速高效酶解。采用核糖核酸酶B作为标准糖蛋白,优化了Pronase E酶切的方案,包括酶切时蛋白与酶的用量比、酶切时间、固定化Pronase E酶的有效贮存时间等;同时优化选择了糖链的富集方法,并对于基质辅助激光解吸飞行时间质谱分析中,糖链适合的基质进行对比选择,从而获得更好的糖链谱图及更为丰富的糖链结构信息。Site-specific elucidation of glycoforms is one of the greatest challenge faced in the structure analysis of glycoproteins. The Pronase E proteolytic enzymes can degrade most of the amide bonds in glycoproteins, while retaining the intact glycans with a small amount of amino acid. When coupled with mass spectrometryor chromatography, the structures of glycoforms can be identified, while the retainingamino acid sequence helped to assign the modification site. In this way, information on site-specific glycoforms can be obtained. The disadvantage of Pronase E digestion is lower digestive efficient, and thus higher concentrations of protease was needed. This study attempts to immobilize Pronase E on a matrix, which resulting in higher local concentration of the enzyme, and enabling fast and efficient digestion of targeted glycoproteins. Using ribonuclease B as a standard glycoprotein, the Pronase E digestion scheme have been optimized, including the ratio of proteinsto enzyme, digestion time and the effective storage time of theimmobilized enzymes. The enrichment methods for the obtained glycans and the matrix selections for matrix-assisted laser desorption time-of-flight mass spectrometry analysis were optimized. The optimized method provided better spectra for glycans and more abundant glycoform information.

关 键 词：糖蛋白 糖链结构 固定化Pronase E 基质辅助激光解吸飞行时间质谱 

分 类 号：Q55[生物学—生物化学] O657.63[理学—分析化学]