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
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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.
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.
引文
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[5] Ma P L, Buschmann M D, Winnik F M. Biomacromolecules, 2010, 11(3): 549
[6] Williams P S. Anal Bioanal Chem, 2016, 408(12): 3247
[7] Zhu C Q, Guo S, Gaoyang Y Y, et al. Chinese Pharmaceutical Journal, 2016, 51(16): 1364 朱尘琪, 郭爽, 高杨亚雅, 等. 中国药学杂志, 2016, 51(16): 1364
[8] Li H X, Mao X H. Progress in Microbiology and Immunology, 2007, 35(1): 54 李海侠, 毛旭虎. 微生物学免疫学进展, 2007, 35(1): 54
[9] Hofmann J, Stuckmann A, Crispin M, et al. Anal Chem, 2017, 89(4): 2318
[10] Opuni K F M, Al-Majdoub M, Yefremova Y, et al. Mass Spectrom Rev, 2018, 37(2): 229
[11] Wu Y, Li X, Chen H B, et al. Food Science, 2010, 31(23): 406 武涌, 李欣, 陈红兵, 等. 食品科学, 2010, 31(23): 406
[12] Wang X F. [MS Dissertation]. Qingdao: Ocean University of China, 2008 王晓斐. [硕士学位论文]. 青岛: 中国海洋大学, 2008
[13] Han J X, Chen Y, Ge Y Q. Journal of Chinese Institute of Food Science and Technology, 2016, 16(7): 201 韩建勋, 陈颖, 葛毅强. 中国食品学报, 2016, 16(7): 201
[14] Shanti K N, Martin B M, Nagpal S, et al. J Immunol, 1993, 151(10): 5354
[15] Ayuso R, Lehrer S B, Reese G. Int Arch Allergy Immunol, 2002, 127(1): 27
[16] Ayuso R, Reese G, Leong-Kee S, et al. Int Arch Allergy Imm, 2002, 129(1): 38
[17] Lee J Y, Lim S, Park S, et al. J Chromatogr A, 2013, 1288: 54
[18] Lee J Y, Min H K, Moon M H. Anal Bioanal Chem, 2011, 400(9): 2953
[19] Lee S T, Yang B, Kim J Y, et al. J Chromatogr A, 2015, 1409: 218
[20] Schachermeyer S, Ashby J, Zhong W. J Chromatogr A, 2013, 1295(5): 107
[21] Ashby J, Schachermeyer S, Duan Y, et al. J Chromatogr A, 2014, 1358: 217
[22] Huang H C, Jiang Z F, Zhu H J. Chemistry Bulletin, 2007, 70(7): 501 黄汉昌, 姜招峰, 朱宏吉. 化学通报, 2007, 70(7): 501
[23] Ni G H, Song H L. Food Research and Development, 2002, 23(5): 73 倪国华, 宋焕禄. 食品研究与开发, 2002, 23(5): 73
[24] Liang Q H, Yang Y, Shao B, et al. Chinese Journal of Chromatography, 2018, 36(5): 480 梁启慧, 杨奕, 邵兵, 等. 色谱, 2018, 36(5): 480
[25] Wang J B. [MS Dissertation]. Hangzhou: Zhejiang Gongshang University, 2018 王金宝. [硕士学位论文]. 杭州: 浙江工商大学, 2018