基于Endo-M N175Q糖链转移反应与丙酮富集糖肽的LC-MS分析糖蛋白N-糖链  

LC-MS Analysis on N-glycans in Glycoproteins Based on Transglycosylation Reaction of Endo-M N175Q and Acetone-precipitated Glycopeptide Enrichment

作　　者：陈奕彤 张斯然 张春波[1] 金东日[1] CHEN Yi-tong;ZHANG Si-ran;ZHANG Chun-bo;JIN Dong-ri(Key Laboratory of Nature Resources of Changbai Mountain&Functional Molecular,Ministry of Education,Department of Chemistry,Yanbian University,Yanji 133002,China)

机构地区：[1]延边大学长白山生物资源与功能分子教育部重点实验室,化学系,吉林延吉133002

出　　处：《分析测试学报》2018年第11期1311-1315,共5页Journal of Instrumental Analysis

基　　金：国家自然科学基金资助项目(21365022,21565027)。

摘　　要：基于化学酶标记和丙酮富集糖肽方法,建立了一种可靠、有效、简单的糖蛋白N-糖链分析方法。以唾液酸糖肽(SGP)为模型糖肽,比较了样品中丙酮加入量对SGP的富集效果,最终选择加入样品体积5倍量的丙酮。用丙酮富集经胰蛋白处理的核糖核酸酶B(RNase B)酶解液中的糖肽,以富集分离得到的糖肽(糖基供体)和PDPZ-Boc-Asn-GlcNAc(糖基受体)作为酶反应底物,进行Endo-M N175Q的转糖基反应,得到PDPZBoc-Asn-GlcNAc-N-糖链标记物。采用YMC C18色谱柱为分析柱,10 mmol/L甲酸铵-乙腈为流动相梯度洗脱,经液相色谱-串联质谱(LC-MS)检测得到5种高甘露糖型糖链。结果表明,丙酮可有效地富集大量肽和少量糖肽混合溶液中的糖肽,Endo-M N175Q可将天然糖肽的糖链转移到PDPZ-Boc-Asn-GlcNAc受体上。将该方法应用于胎球蛋白N-糖链分析,检测到5种复杂型N-糖链。该研究为各种糖蛋白N-糖链检测提供了新的分析方法。A reliable,efficient and simple method was developed for the determination of N-glycans in glycoproteins based on the enzymatic labeling and the acetone-precipitated glycopeptides enrichment.Taking sialylglycopeptide(SGP)for instance,a 5-fold volume of acetone was confirmed to be appropriate for the selective precipitation of SGP by comparing the enriching effects of adding volumes of acetone on SGP.To confirm the selectivity of acetone precipitation for N-glycopeptides,tryptic digest from bovine ribonuclease B(RNase B)was treated with acetone,following by chemoenzymatic labeling and a transglycosylation reaction utilizing Endo-M N175Q and PDPZ-Boc-Asn-GlcNAc as acceptor,and the PDPZ-Boc-Asn-GlcNAc-N-glycans were obtained.The chromatographic separation was conducted on a YMC C 18 column using water containing 10 mmol/L HCOONH 4-acetonitrile as mobile phase by gradient elution.The analysis was performed by ESI-MS in positive mode.Five high mannose types of N-glycans in RNase B were detected.Results showed that N-glycosylated peptides could be enriched in the precipitate by adding acetone,and the chemoenzymatic labeling was performed in the presence of the glycopeptides and PDPZ-Boc-Asn-GlcNAc as enzymic catalytic substrates.The method was successful in acquiring the comprehensive MS data for five complex types of glycans in fetuin.Therefore,it is a powerful tool for the determination of N-glycans in glycoproteins.

关 键 词：糖蛋白 N-糖链 酶标记 液相色谱-质谱(LC-MS) 

分 类 号：O657.63[理学—分析化学] O629.73[理学—化学]