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预防医学  2021, Vol. 33 Issue (8): 768-771    DOI: 10.19485/j.cnki.issn2096-5087.2021.08.003
  论著 本期目录 | 过刊浏览 | 高级检索 |
虾过敏原蛋白质谱分析方法的酶解条件优化研究
潘晓东, 黄百芬, 蔡增轩, 王梦莉, 韩见龙, 陈苘
浙江省疾病预防控制中心理化毒理所,浙江 杭州 310051
Optimization of enzymatic digestion conditions of shrimp allergens in mass spectrometry by response surface methodology
PAN Xiaodong, HUANG Baifen, CAI Zengxuan, WANG Mengli, HAN Jianlong, CHEN Qing
Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang 310051, China
全文: PDF(1138 KB)  
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摘要 目的 优化虾过敏原蛋白质谱分析试验的胰蛋白酶酶解条件,提高质谱对虾过敏原蛋白的分析能力。方法 采用响应面试验设计对虾过敏原蛋白酶酶解的pH值、温度和时间进行优化,用胰蛋白酶消化肽段,经色谱分离后采用四极杆静电场轨道离子阱高分辨质谱测定,利用UniProt蛋白数据库和MaxQuant软件对过敏原蛋白进行鉴定和定量分析。结果 获得虾中2种过敏原蛋白,分别为原肌球蛋白和精氨酸激酶,质谱强度为100.2×106~436.5×106。响应面分析结果显示,酶解时间为4.29 h、温度为44.15 ℃、溶液pH值为6.55时,过敏原蛋白的质谱响应强度最高,为457.48×106。采用溶液pH值6.5、温度44 ℃、时间4.2 h进行验证试验,获得过敏原蛋白的平均响应强度为448.1×106,与预测值偏差为2.1%。结论 采用响应面法可以优化蛋白酶酶解条件,当pH值为6.5、温度为44 ℃、时间为4.2 h时具有最佳的酶解能力。
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潘晓东
黄百芬
蔡增轩
王梦莉
韩见龙
陈苘
关键词 过敏原蛋白响应面酶解质谱    
AbstractObjective To optimize the enzymatic digestion conditions of trypsin, so as to improve the testing capacity of mass spectrometry for shrimp allergens. Methods The enzymatic digestion test was carried out by response surface methodology for optimizing pH, temperature and time. After enzymatic hydrolysis, the peptides were separated by chromatography and determined by high-resolution mass spectrometry with Q-orbitrap. The allergen protein was identified and quantified by UniProt database and MaxQuant software. Results Two allergen proteins, tropomyosin and arginine kinase, were isolated from shrimp, and their intensities ranged from 100.2×106 to 436.5×106. Response surface analysis showed that when the digestion time was 4.29 hours, the temperature was 44.15 ℃ and pH value was 6.55, the maximal intensity of the allergen proteins was 457.48×106. The experiment was validated with the digestion time of 4.2 h, pH value of 6.5, and temperature of 44 ℃, then resulted in the average intensity of 448.1×106. The deviation from the predicted value was 2.1%. Conclusion s The conditions of enzymatic digestion can be optimized by response surface methodology. The enzyme may have the best performance with the pH value of 6.5, temperature of 44 ℃ and digestion time of 4.2 hours.
Key wordsallergen protein    response surface methodology    enzymatic digestion    mass spectrometry
收稿日期: 2021-03-12      出版日期: 2021-08-10
ZTFLH:  R392  
基金资助:浙江省医药卫生科技计划项目(2019316301)
通信作者: 陈苘,E-mail:qingchen@cdc.zj.cn   
作者简介: 潘晓东,博士,副研究员,主要从事理化分析工作
引用本文:   
潘晓东, 黄百芬, 蔡增轩, 王梦莉, 韩见龙, 陈苘. 虾过敏原蛋白质谱分析方法的酶解条件优化研究[J]. 预防医学, 2021, 33(8): 768-771.
PAN Xiaodong, HUANG Baifen, CAI Zengxuan, WANG Mengli, HAN Jianlong, CHEN Qing. Optimization of enzymatic digestion conditions of shrimp allergens in mass spectrometry by response surface methodology. Preventive Medicine, 2021, 33(8): 768-771.
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http://www.zjyfyxzz.com/CN/10.19485/j.cnki.issn2096-5087.2021.08.003      或      http://www.zjyfyxzz.com/CN/Y2021/V33/I8/768
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