Abstract：Objective To establish the ultra-high performance liquid chromatography coupled with quadrupole orbitrap mass spectrometry ( UPLC-Q-Orbitrap-MS ) for the analysis of allergen protein in Macrobrachium. Methods Based on the strategy of bottom-up protein analysis, the proteins in Macrobrachium samples were extracted by Tris-HCl, digested by trypsin at 40 ℃ for 6 hours, separated by chromatography, and analyzed by Q-Orbitrap-MS spectrometry ( Full MS/dd-MS2, TopN=10 ) . Allergen proteins were identified with UniProt protein database and Proteome Discoverer software. Results Four kinds of allergen proteins were obtained, which were tropomyosin, arginine kinase, sarcoplasmic calcium binding protein and hemocyanin. The coverage rates of peptides in proteins were 53%, 36%, 12% and 12%, respectively. Post translation modifications were methylation of aspartic acid (D), deacylylation of aspartic acid ( N ) , glutamyl ammonia ( Q ) and oxidation of methionine ( M ) . Conclusions The UPLC-Q-Orbitrap-MS can identified abundant peptide and fragment information with high sensitivity and resolution, which provides a technical support for the analysis of shrimp allergens.
潘晓东, 黄百芬, 蔡增轩, 王梦莉, 韩见龙, 陈苘. 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.
PAN Xiaodong, HUANG Baifen, CAI Zengxuan, WANG Mengli, HAN Jianlong, CHEN Qing. Analysis of allergen protein in Macrobrachium by ultra-high performanceliquid chromatography coupled with quadrupole orbitrap mass spectrometry. Preventive Medicine, 2020, 32(10): 1010-1012.
 陈红兵. 食物过敏研究新动态[J].食品安全质量检测学报,2017,8(4):1085-1086.  YU W,FREELAND D M H,NADEAU K C. Food allergy:immune mechanisms,diagnosis and immunotherapy[J]. Nat Rev Immunol,2016,16(12):751-765.  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.  WAI C Y,LEUNG N Y,LEUNG P S,et al.Immunotherapy of food allergy:a comprehensive review[J].Clin Rev Allergy Immunol,2019,57(1):55-73.  PRESTER L.Seafood allergy,toxicity,and intolerance:a review[J].J Am Coll Nutr,2016,35(3):271-283.  RUETHERS T,TAKI A C,JOHNSTON E B,et al.Seafood allergy:a comprehensive review of fish and shellfish allergens[J].Mol Immunol,2018,100:28-57.  FAISAL M,VASILJEVIC T,DONKOR O N.A review on methodologies for extraction,identification and quantification of allergenic proteins in prawns[J].Food Res Int,2019,121:307-318.  MARZANO V,TILOCCA B,FIOCCHI A G,et al.Perusal of food allergens analysis by mass spectrometry-based proteomics[J].J Proteomics,2020,215:103636.  CHEN Q,PAN X D,HUANG B F,et al.Screening of multi-class antibiotics in pork meat by LC-Orbitrap-MS with modified QuEChERS extraction[J].RSC Advances,2019,9(48):28119-28125.  张伟. 定量蛋白质组学质谱采集技术进展[J]. 分析化学,2014,42(12):1859-1868.  张晓艺,张秀尧,蔡欣欣,等.超高效液相色谱联用三重四极杆质谱法同时测定蜂蜜中氯霉素、甲硝唑和林可霉素[J].预防医学,2019,31(2):212-216.  李紫薇,张晓梅,胡玲萍,等.南极磷虾过敏原的超高效液相色谱-飞行时间质谱方法检测[J].现代食品科技,2018,34(9):222-227.  熊岑,李苑雯,郑彦婕,等. 静电场轨道阱质谱分析技术在食品分析中的应用进展[J]. 食品科学,2015,36(13):283-287.  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.  FABER M A,PASCAL M,El KHARBOUCHI O,et al.Shellfish allergens:tropomyosin and beyond[J].Allergy,2017,72(6):842-848.  LOPATA A L,KLEINE-TEBBE J,KAMATH S D.Allergens and molecular diagnostics of shellfish allergy:part 22 of the Series Molecular Allergology[J]. Allergo J Int,2016,25(7):210-218.  LINDA M,ELISABETTA D A,NICOLA M,et al.Comprehensive overview and recent advances in proteomics MS based methods for food allergens analysis[J].TrAC Trends Anal Chem,2018,106:21-36.  傅玲琳,梁亚云,赵淑淑,等.中国对虾蛋白致敏小鼠模型构建及主要过敏原的双向电泳联合质谱鉴定[J].中国食品学报,2017,17(12):172-181.  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.