β-伴大豆球蛋白的N-糖基化位点及其N-糖链的质谱分析  

Mass Spectrometric Analysis of N-Glycosylation Sites and N-Glycans of β-Conglycinin

作　　者：李玲梅 王承健 韩健利 柳人丹 温娅楠 魏明 晋万军 黄琳娟[1] 王仲孚[1] 

机构地区：[1]西北大学生命科学学院,西部资源生物与现代生物技术教育部重点实验室,西安710069

出　　处：《高等学校化学学报》2018年第3期427-434,共8页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金(批准号：31670808,31600647,31370804); 陕西省教育厅自然科学专项(批准号：16JK1782); 陕西省重点实验室项目基金(批准号：16JS109); 西北大学校内基金(批准号：15NW18)资助~~

摘　　要：以β-伴大豆球蛋白(β-conglycinin)为研究对象,利用Trypsin和Pepsin酶对其进行水解,以Con A亲和层析柱富集纯化糖肽,并用糖苷酶PNGase F和Endo H分别酶解糖肽,再采用电喷雾质谱(ESI-MS)和串联质谱(MS/MS)对所得肽段的氨基酸序列及糖链的结构进行了分析,最后通过数据库检索对分析结果进行验证.结果表明,β-conglycinin具有5个N-糖基化位点,分别为α亚基的199和455位天冬酰胺(Asn),α'亚基的215和489位Asn及β亚基的326位Asn,且每个糖基化位点均被H5N2,H6N2,H7N2和H8N2这4种高甘露糖型N-糖链所修饰.本研究为各种糖蛋白的糖基化位点及其对应的糖链结构的鉴定分析提供了方法参考,并为深入理解大豆糖蛋白抗原表位的特异性及致敏机理提供了依据.As one of common allergenic food,soybean makes people allergic attributed to it＇s allergenβ-conglycinin,of which the analysis of glycosylation sites and N-glycan structure has a significant meaning for understanding antigenic properties and sensitization mechanism of soybean in-depth.However,due to the limitations of analytical techniques,the corresponding relationship between glycosylation sites and N-glycan structure is lack of comprehensive understanding.In this study,β-conglycinin was hydrolyzed into peptide fragments using Trypsin and Pepsin,followed by enrichment of glycopeptides using Con A affinity chromatography.Subsequently,two aliquots of the glycopeptide samples were subjected to digestion with PNGase F and Endo H,respectively.The recovered peptides and glycans were analyzed in detail by means of electrospray ionization mass spectrometry（ESI-MS） and tandem mass spectrometry（MS/MS）.The obtained data of peptides were retrieved using the Mascot software.These results show that β-conglycinin has 5 N-glycosylation sites,including the 199 th and 455 th Asn residues of the α subunit,the 215 th and 489 th Asn residues of theα＇ subunit and the 326 th Asn residue of the β subunit,and each glycosylation site was modified by the four high-mannose type N-glycans,including H5N2,H6N2,H7N2 and H8N2.This study provides a new strategy for the identification of glycosylation sites and corresponding glycan structures of various glycoproteins and offers a structural basis for deep understanding of the specificity of soybean protein glycoprotein epitopes and allergic mechanism.

关 键 词：Β-伴大豆球蛋白 糖基化位点 N-糖链 质谱 

分 类 号：O657.6[理学—分析化学] O629.12[理学—化学]