摘要
建立了基于特征肽段检测虾制品中过敏原蛋白的胰酶酶解/高效液相色谱-四极杆-飞行时间质谱方法。采用6 mol/L盐酸胍和50 mmol/L三羟甲基氨基甲烷-盐酸(Tris-HCl)缓冲液各700μL分别作为蛋白质的变性提取试剂,通过优化酶解时间和特征肽段,确定采用50 mmol/L二硫苏糖醇、100 mmol/L碘乙酰胺破坏并抑制二硫键的形成,而后在100μL 500 mmol/L碳酸氢铵弱碱性环境中经10 mg/mL胰蛋白酶酶解,以乙腈和0.1%甲酸水溶液为流动相,采用Caprisil C_(18)-X(100 mm×2.1 mm,1.8μm)色谱柱进行分离,电喷雾双喷离子源(Dual AJS ESI)正离子模式下电离,利用四极杆-飞行时间质谱通过Auto MS/MS采集离子迭代排除的方法循环采样。根据肽段出峰情况,选择IQLLEEDLER作为原肌球蛋白的特征肽段,利用外标法定量。结果证明,实验选取的特征肽段在线性范围内线性关系良好,相关系数(r^(2))为0.9920,检出限为0.68μg/L,定量下限为2.28μg/L,在2、10 mg/kg加标水平下样品的回收率为84.5%~101%。该方法前处理简单、灵敏度高、重现性好、定性定量准确,可为虾过敏原的检验鉴定提供技术支持,并为基于蛋白质特征肽段的动物源成分识别提供方法参考。
In this study,an analytical method was established for determination of allergenic protein in shrimp products based on characteristic peptides by pancreatic enzymolysis/high performance liquid chromatography-quadrupole time of flight mass spectrometry.With 100μL 6 mol/L guanidine hydrochloride and 700μL 50 mmol/L Tris-HCl buffer as denaturating and extraction reagents,50 mmol/L dithiositol and 100 mmol/L iodoacetamide were used to destroy and inhibit the formation of disulfide bonds by optimizing the enzymolysis time and the selection of characteristic peptide.After enzymolysis with 10 mg/mL trypsin in a weak alkaline environment created by 100μL and 500 mol/L ammonium bicarbonate,the analytes were separated by using a Caprisil C_(18)-X(100 mm×2.1 mm,1.8μm)column with 0.1%formic acid aqueous solution and acetonitrile as the mobile phase for elution.The ionization was performed by Dual AJS ESI in positive mode,then the ions were detected by quadrupole time of flight mass spectrometry,with cyclic sampling performed by the Auto MS/MS ion acquisition iterative exclusion mode.IQLLEEDLER was selected as the characteristic peptide of tropomyosin according to chromatographic results.The results showed that the characteristic peptides selected in the experiment had good linear relationship in the linear range,the correlation coefficient(r^(2))was 0.9920,the limit of detection was 0.68μg/L,and the limit of quantitation was 2.28μg/L.The recoveries of the samples were 84.5%-101%at 2 and 10 mg/kg standard levels.The method is simple,sensitive,reproducible and accurate,which can provides technical support for the identification of shrimp allergens and method reference for the identification of animal-derived components based on characteristic peptides.
作者
史洪飞
周修齐
徐伯芃
周用武
徐成鑫
周亦飞
冶金
张吕悦
刘柯宇
郭明鑫
SHI Hong-fei;ZHOU Xiu-qi;XU Bo-peng;ZHOU Yong-wu;XU Cheng-xin;ZHOU Yi-fei;YE Jin;ZHANG Lü-yue;LIU Ke-yu;GUO Ming-xin(Key Laboratory of State Forestry and Grassland Administration on Wildlife Evidence Technology,College of Criminal Science and Technology,Nanjing Police University,Nanjing 210023,China;Dagang Customs Anti-Smuggling Branch,Qingdao Customs Anti-Smuggling Bureau,Qingdao 266005,China)
出处
《分析测试学报》
北大核心
2025年第10期2162-2168,共7页
Journal of Instrumental Analysis
基金
国家重点研发计划项目(2023YFC3304000)
2025年省级大学生创新重点项目(X202512263052)。
关键词
胰酶酶解
高效液相色谱-四极杆-飞行时间质谱法
虾制品
过敏原蛋白
特征肽段
pancreatic enzymolysis
high performance liquid chromatography-quadrupole-time of flight mass spectrometry
shrimp products
allergenic protein
characteristic peptide segments
