Mass Spectrometric Analysis of N-Glycoforms of Soybean Allergenic Glycoproteins Separated by SDS-PAGE.
Mass Spectrometric Analysis of N-Glycoforms of Soybean Allergenic Glycoproteins Separated by SDS-PAGE.
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DOI：
10.1021/acs.jafc.6b02773
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发表时间：
2016-09
影响因子：
6.1
通讯作者：
Lingmei Li;Chengjian Wang;Shan Qiang;Jixiang Zhao;Shuang Song;W. Jin;Bo Wang;Ying Zhang;Linjuan Huang;Zhongfu Wang
中科院分区：
文献类型：
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作者：
Lingmei Li;Chengjian Wang;Shan Qiang;Jixiang Zhao;Shuang Song;W. Jin;Bo Wang;Ying Zhang;Linjuan Huang;Zhongfu Wang
许多蛋白质的糖基化与食物过敏密切相关，筛选和分析相关糖蛋白和糖过敏原是这一领域研究的基础。在此，我们描述了详细的N-糖型分析的所有糖蛋白组分的大豆分离蛋白（SPI）分离的十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），以揭示更多的大豆糖蛋白的聚糖部分的结构特征。采用SDS-PAGE对SPI进行分离，回收蛋白条带，采用本课题组新开发的一锅法进行胶内N-聚糖释放和标记，然后采用电喷雾质谱（ESI-MS）和在线亲水作用液相色谱-电喷雾串联质谱（HILIC-ESI-MS/MS）进行详细分析。结果，我们发现7条条带主要含有低聚甘露糖型聚糖; 2条主要含有核心α 1，3-岩藻糖基化聚糖，6条不含聚糖。本研究首次在条带1、2和6中发现核心α 1，3-岩藻糖基化N-聚糖，并将条带3、4、5和7公开为糖蛋白及其N-糖型。因此，本研究可以扩展对大豆蛋白糖基化的认识，为大豆过敏原的研究提供重要的结构参考。
Glycosylation of many proteins has been revealed to be closely related with food allergies, and screening and structural analysis of related glycoproteins and glycoallergens are essential for studies in this field. Herein, we describe detailed N-glycoform analysis of all glycoprotein fractions of soybean protein isolate (SPI) separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to disclose structural features of the glycan moieties of more soybean glycoproteins. SPI was fractionated by SDS-PAGE, and the generated protein bands were recovered and subjected to in-gel N-glycan release and labeling using a one-pot method newly developed by our group, followed by detailed analysis by electrospray ionization mass spectrometry (ESI-MS) and online hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS). As a result, we found seven bands mainly containing oligomannose-type glycans; two mainly contain core α1,3-fucosylated glycans, and six have no glycans. This study is the first report that discovers core α1,3-fucosylated N-glycans in bands 1, 2, and 6 and discloses bands 3, 4, 5, and 7 as glycoproteins and their N-glycoforms. Therefore, it can expand our knowledge about soybean protein glycosylation and provide significant structural reference for research of soybean allergens.