非对称流场流分离-超高效液相色谱-串联四极杆飞行时间质谱用于过敏原蛋白表位筛选  

Asymmetrical flow field flow fractionation combined with ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry used to screen allergen protein epitopes

作　　者：李阳 杨奕[2] 邵兵[2] 邹悦 宋宇[1] 舒琳 梁启慧[1] 韩南银[1] LI Yang;YANG Yi;SHAO Bing;ZOU Yue;SONG Yu;SHU Lin;LIANG Qihui;HAN Nanyin(School of Pharmaceutical Sciences,Peking University,Beijing 100191,China;Beijing Center for Disease Prevention and Control,Beijing 100013,China)

机构地区：[1]北京大学药学院,北京100191 [2]北京市疾病预防控制中心,北京100013

出　　处：《色谱》2019年第4期398-403,共6页Chinese Journal of Chromatography

基　　金：北京市自然科学基金(7162088);北京市科技计划(Z181100009318009)~~

摘　　要：应用非对称流场流分离(AF4)技术结合超高效液相色谱-四极杆飞行时间质谱(UPLC-QTOF-MS)对过敏原蛋白表位进行筛选。将选择的过敏原蛋白(虾原肌球蛋白,TM)酶解后经UPLC-QTOF-MS分析,建立蛋白质肽谱。将TM酶解后的肽段与免疫球蛋白E混合孵育30 min,孵育过程中含有抗原表位的特异性肽段与免疫球蛋白E(IgE)结合,未结合的肽段仍留在溶液中。将孵育后的溶液进行AF4分离,已结合的肽段随IgE一起由出口流出,未结合的肽段透过分离通道膜,滤出至废液。收集出口流出的组分进行UPLC-QTOF-MS分析,与蛋白质肽谱匹配,找到特异性肽段,进而检测抗原表位。本研究扩展了非对称流场流分离技术的应用,对过敏原蛋白表位的检测进行了初步探索,为过敏原蛋白表位的研究提供了一种新的研究策略。Asymmetrical flow field flow fractionation (AF4) combined with ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to screen allergen protein epitopes. The selected allergen protein (tropomyosin, TM) was enzymatically digested into peptide segments and analyzed via UPLC-QTOF-MS to establish a protein-specific peptide database. The peptide segments were incubated with immunoglobulin E (IgE) for 30 min. During the incubation procedure, the specific peptide segments (with the antigen epitope) combine with IgE while the other peptide segments remain in solution. After incubation , the solution was injected into the AF4 device. The combined peptide segments flowed out of the outlet along with IgE, and the other peptide segments flowed into the waste liquid. The components of outlet were then collected, analyzed by UPLC-QTOF-MS, and the results matched with the spectra of the protein peptides. Eventually the specific peptide segments were identified to detect the antigen epitopes. This study extends the application of AF4 with a preliminary exploration of the detection of an allergen protein epitope, providing a novel research strategy for the screening of allergen epitopes.

关 键 词：非对称流场流分离 质谱 过敏原 表位 免疫球蛋白E 

分 类 号：O658[理学—分析化学]