Abstract：Objective 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.
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.
 刘晓宇,李荔,刘志刚.食物过敏原介导食物过敏机制研究进展[J].食品安全质量检测学报,2012,3(4):235-239.  CHEN Q,PAN X D,HUANG B F,et al.Authentication of shrimp muscle in complex foodstuff by in-solution digestion and high-resolution mass spectrometry[J].Rsc Advances,2017,7(52):32903-32908.  潘晓东,黄百芬,蔡增轩,等.超高效液相色谱-四极杆静电场轨道离子阱质谱分析沼虾过敏原蛋白[J].预防医学,2020,32(10):1010-1012,1017.  NOOR Z,AHN S B,BAKER M S,et al.Mass spectrometry-based protein identification in proteomics-a review[J].Brief Bioinform,2021,22(2):1620-1638.  AEBERSOLD R,MANN M.Mass-spectrometric exploration of proteome structure and function[J].Nature,2016,537(7620):347-355.  SWITZAR L,GIERA M,NIESSEN W M A.Protein digestion: an overview of the available techniques and recent developments[J]. J Proteome Res,2013,12(3):1067-1077.  CHELLADURAI S J S,MURUGAN K,RAY A P,et al.Optimization of process parameters using response surface methodology: a review[J]. Mater Today Proc,2021,37:1301-1304.  徐长明,张纪阳,张伟,等. 应用于Bottom-up 蛋白质鉴定的质谱数据采集策略研究进展[J].分析化学,2013,41(7):1120-1128.  MONACI L,DE ANGELIS E,MONTEMURRO N,et al.Comprehensive overview and recent advances in proteomics MS based methods for food allergens analysis[J].TrAC,2018,106:21-36.  VIDOVA V,SPACIL Z.A review on mass spectrometry-based quantitative proteomics:targeted and data independent acquisition[J].Anal Chim Acta,2017,964:7-23.  SINITCYN P,RUDOLPH J D,COX J.Computational methods for understanding mass spectrometry-based shotgun proteomics data[J].Annu Rev Biomed Data Sci,2018,1:207-234.  DUPREE E J,JAYTHIRTHA M,YORKEY H,et al.A critical review of bottom-up proteomics:the good, the bad,the future of this field[J/OL].Proteomes,2020,8(3)[2021-05-26].https://pubmed.ncbi.nlm.nih.gov/32640657/ .DOI: 10.3390/proteomes8030014.  潘晓东,黄百芬,张京顺,等.猪肉中16种β-内酰胺类抗生素UPLC-Q-Orbitrap检测方法研究[J].预防医学,2019,31(11):1100-1104.  YOLMEH M,JAFRI S M.Applications of response surface methodology in the food industry processes[J].Food Bioproc Tech,2017,10(3):413-433.  周志昊,王凯杰,龚萃萍,等.响应面法优化紫绒丝膜菌产紫色素培养基[J].中国食品添加剂,2016(10):77-82.