基于特征肽段结合超高效液相色谱-串联质谱法的乳制品真实性鉴别方法研究

Study on the authenticity identification of dairy products based on characteristic peptides combined with ultra performance liquid chromatography-tandem mass spectrometry

中文关键词:  功性能乳蛋白  靶向蛋白质组学  超高效液相色谱-四极杆-静电场轨道阱高分辨质谱法  超高效液相色谱-串联质谱法  鉴别

英文关键词:functional proteins  targeted proteomics  ultra performance liquid chromatography-triple quadrupole- Orbitrap high resolution mass spectrometry  ultra performance liquid chromatography-tandem mass spectrometry  authenticity identification

中文摘要:

      目的 基于超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)建立乳制品中乳铁蛋白、乳桥蛋白、奶牛A1 β-酪蛋白和A2 β-酪蛋白、水牛A2 β-酪蛋白的检测方法, 开展市售乳制品中功能性蛋白质检测和乳制品真实性鉴评分析。方法 乳蛋白经酶解后, 应用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱法(ultra performance liquid chromatography-triple quadrupole-Orbitrap high resolution mass spectrometry, UPLC-Q/Orbitrap HRMS)和UPLC-MS/MS筛选获得可用于定性定量分析的特征肽段, 基于UPLC-MS/MS建立同时检测LF、LPN、奶牛A1 β-CN、奶牛A2 β-CN和水牛A2 β-CN的方法并应用于乳制品检测。结果 5种待测蛋白在相应的浓度范围内线性关系良好(r2>0.99), 加标回收率在82.1%~105.5%之间, 相对标准偏差均小于5%, 78份乳制品的检测结果表明同类样品LF含量差异较大, 不同类液体乳中LPN均值差异不大。结论 本方法性能良好, 适用于乳制品中乳桥蛋白等功能性蛋白的同时检测, 也可以实现乳制品中乳蛋白物种来源的鉴别。

英文摘要:

      Objective To develop a method for the detection of lactoferrin, lactopontin, A1 β-casein and A2 β-casein from cows, and A2 β-casein from buffalo in dairy products based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and conduct functional protein testing and identification analysis of commercially available dairy products. Methods After enzymatic digestion of milk proteins, ultra performance liquid chromatography-triple quadrupole-Orbitrap high resolution mass spectrometry (UPLC-Q/Orbitrap HRMS) and UPLC-MS/MS were applied to screen and obtain specific peptides for qualitative and quantitative analysis, and a method for the simultaneous detection of lactoferrin, lactopontin, A1 β-casein and A2 β-casein from cows, and A2 β-casein from buffalo based on UPLC-MS/MS was developed and applied to dairy products detection. Results The 5 kinds of proteins to be measured showed good linearity (r2>0.99) in the corresponding concentration ranges, and the spiked recoveries ranged from 82.1% to 105.5% with the relative standard deviations less than 5%. The results of 78 dairy products showed that the LF content of similar samples varied widely, and the mean LPN values in different types of liquid milk did not differ significantly. Conclusion This method has good performance and is suitable for the simultaneous detection of functional proteins such as lactoferrin in dairy products, and also for the identification of the origin of milk protein species in dairy products.