微量生物样品中糖蛋白N-糖链电喷雾电离质谱分析前处理方法的建立  

Pretreatment Method for Detecting of N-linked Glycans of Glycoprotein in Micro-scale Complex Biological Sample based on Electrospray Ionization Mass Spectrometry

作　　者：张英[1] 刘影[1] 刘洋[1] 强珊[1] 杨冰莹[1] 黄琳娟[1] 王仲孚[1] 

机构地区：[1]西北大学生命科学学院,西安710069

出　　处：《分析化学》2016年第2期265-272,共8页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(Nos.31170773;31370804;31300678)资助~~

摘　　要：基于电喷雾电离质谱检测技术,建立了一种可靠、高效、简单,适合于微量糖蛋白N-糖链解离、富集纯化的方法。以糖蛋白牛胰核糖核酸酶(Rib B)和卵清白蛋白(OVA)为模型蛋白直接酶解,比较了4种方法纯化酶解样品的效果。比较了直接酶解和经过聚偏氟乙烯(PVDF)膜富集后酶解微量复杂生物来源样品胎牛血清的酶解效果,最终建立了微量生物样品中糖蛋白N-糖链的质谱分析前处理方法。采用PVDF膜吸附复杂生物样品中的糖蛋白,N-糖苷酶F(PNGase F)酶直接在膜上完成糖链释放(37℃,24 h),采用微晶纤维素柱结合石墨碳柱对糖链进行富集纯化,用于微克级胎牛血清和健康人血清中N-糖链质谱分析的前处理。本方法通用性好,在微量生物样品糖链质谱分析检测的前处理方面具有一定应用价值。A reliable, efficient and simple method was developed for releasing, enrichment and purification of N-glyean from glycoprotein based on electrospray ionization mass spectrometric （ESI-MS） detection technology. The bovine ribonuclease B （Rib B） and ovalbumin were used as model glycoprotein samples and subjected to direct enzymolysis to investigate the purification effects of four different purification methods. Then the enzymatic efficiency of micro-scale complex fetal bovine serum sample subjected to direct and indirect enzymolysis after polyvinylidene fluoride （PVDF） membrane enrichment was fully investigated. Finally, a pretreatment method of N-linked glycans from glyeoprotein in micro-scale complex biological sample analyzed by ESI-MS was established. The PNGase F was exploited for directly releasing of glycans from glyeoprotein enriched on PVDF membrane （37~C, 24 h）. Then microcrystalline cellulose chromatography column combined with graphite carbon chromatography column was used to enrich and purify glycans from enzymolysis products. The assay was eventually applied to pretreatment of N-glycan in micro-scale biological samples （ μg level） of fetal bovine serum and healthy human serum for MS analysis. The method with universality showed good prospects in pretreatment of N-glycan in micro-scale biological sample for MS analysis.

关 键 词：微量分析 糖蛋白 N-糖链 电喷雾质谱 前处理 

分 类 号：Q503[生物学—生物化学] O657.63[理学—分析化学]