Li Lingmei, Wang Chengjian, Qiang Shan, Zhao Jixiang, Song Shuang, Jin Wanjun, Wang Bo, Zhang Ying, Huang Linjuan, Wang Zhongfu
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University , Xi'an 710069, China.
School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University , Dalian 116034, China.
J Agric Food Chem. 2016 Oct 5;64(39):7367-7376. doi: 10.1021/acs.jafc.6b02773. Epub 2016 Sep 21.
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.
许多蛋白质的糖基化已被揭示与食物过敏密切相关，对相关糖蛋白和糖过敏原进行筛选及结构分析是该领域研究的关键。在此，我们详细描述了通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分离的大豆分离蛋白（SPI）所有糖蛋白组分的N-糖型分析，以揭示更多大豆糖蛋白聚糖部分的结构特征。通过SDS-PAGE对SPI进行分级分离，回收产生的蛋白条带，并使用我们团队新开发的一锅法进行胶内N-聚糖释放和标记，随后通过电喷雾电离质谱（ESI-MS）以及在线亲水相互作用液相色谱与电喷雾电离串联质谱联用（HILIC-ESI-MS/MS）进行详细分析。结果，我们发现七条主要含有低聚甘露糖型聚糖的条带；两条主要含有核心α1,3-岩藻糖基化聚糖，还有六条不含聚糖。本研究首次报道在第1、2和6条带中发现核心α1,3-岩藻糖基化N-聚糖，并揭示第3、4、5和7条带为糖蛋白及其N-糖型。因此，它可以拓展我们对大豆蛋白糖基化的认识，并为大豆过敏原研究提供重要的结构参考。
