超高效液相色谱-串联质谱法鉴定驼乳及其制品中的动物源性成分

Identifying animal-derived components in camel milk and its products by ultra-high performance liquid chromatography-tandem mass spectrometry

采用蛋白质组学技术建立了驼乳及其制品中的动物乳源性成分特异性肽生物标志物的鉴定方法。样品经脱脂、蛋白质提取、胰蛋白酶水解后,利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱仪(UHPLC-Q/Exactive-HRMS)和Protein Pilot软件,实现了多肽生物标记物的鉴定;然后通过基本局部比对搜索工具(BLAST)与Uniprot数据库对比分析,筛选出了骆驼、家牛、水牛、牦牛、山羊、绵羊、驴和马共8个物种的22条肽生物标志物;最后利用超高效液相色谱-三重四极杆质谱(UHPLC-QqQ-MS)系统对这22条特征性多肽进行验证,采用多反应监测(MRM)模式建立了定量方法。实验优化了骆驼奶中酪蛋白的预处理方法,如冷冻脱脂、沉淀蛋白试剂和复溶液的选择等,并建立了基于生物标志物肽测定牛和山羊奶/奶粉掺假骆驼奶/奶粉的无标记定量方法。将牛、山羊和骆驼奶/奶粉等比例混合,分别对牛和山羊的特征肽段进行检测,结果显示,该方法对掺假液体乳/固体奶粉样品显示出良好的线性关系,抗干扰能力强,灵敏度高,重复性好,液态奶和奶粉的掺杂结果均与理论值接近。另外,该方法还被应用于11种骆驼奶和奶粉中家牛、水牛、牦牛、山羊、绵羊、驴和马乳源性成分的鉴别,为骆驼乳及乳制品中多种动物乳源性成分的鉴别提供了有效的技术平台。

A method for identifying specific peptide biomarkers of animal-milk-derived components in camel milk and its products was established using proteomics.Samples were prepared by defatting,protein extraction,and trypsin hydrolysis,and proteins and peptides were identified using ultra-high performance liquid chromatography-quadrupole/electrostatic orbitrap-high resolution mass spectrometry(UHPLC-Q/Exactive-HRMS)and Protein Pilot software.Twenty two peptide biomarkers from eight species(i.e.,Camelus,Bos taurus,Bubalus bubalis,Bos grunniens/Bos mutus,Capra hircus,Ovis aries,Equus asinus,Equus caballus)were identified by comparing the basic local alignment search tool(BLAST)with the Uniprot database.Verification of these marker peptides were performed quantitatively using a UHPLC-triple-quadrupole mass-spectrometry(QqQ-MS)system by multiple reaction monitoring(MRM).The pretreatment method of casein in camel milk was optimized,such as defatting,protein precipitation,and re-dissolving buffer solution.The effects of various mass-spectrometry parameters,such as atomization gas,heating-and drying-gas flow rates,and desolvation-tube(DL)and ion-source-interface temperatures on ion-response intensity were optimized.Camel milk signature peptides were detected in a mixture of milk from other seven species to ensure specificity for the selected biomarker peptides.The signature peptides of seven other species were also detected in camel milk.No mutual interference between the selected biomarker peptides of the various species was observed.Adulterated camel milk and milk powder were also quantitatively studied by adding 0,2.5%,5%,10%,25%,50%,75%,and 100% bovine milk or goat milk to camel milk.Similarly,the same mass proportion of bovine milk powder or goat milk powder was added to camel milk powder.A quantitative standard curve for adulteration was constructed by plotting the peak areas of characteristic cow or goat peptide segments in each mixed sample against the mass percentage of the added adulterant.The adulteration standard curves exhibited good linearity,with correlation coefficients(r^(2))greater than 0.99.The limits of detection and quantification(LODs and LOQs,respectively)of the method were determined as three-and ten-times the signal-to-noise ratio(S/N).The minimum adulteration LODs of bovine milk and goat milk in camel milk were determined to be 0.35% and 0.49%,respectively,and the minimum LOQs were 1.20% and 1.69%,respectively.The minimum adulteration LODs of bovine milk powder and goat milk powder in camel milk powder were determined to be 0.68% and 0.73%,respectively,and the minimum LOQs were 1.65%and 2.45%,respectively.The accuracy of the adulteration quantification method was investigated by validating the quantitative detection results for 1∶1∶1(mass ratio)mixtures of camel milk,bovine milk,and goat milk,as well as camel-milk powder,bovine milk powder,and goat-milk powder,which revealed that this method exhibits good linearity,strong anti-interference,high sensitivity,and good repeatability for adulterated liquid-milk/solid-milk-powder samples.The adulteration results for both liquid milk and milk powder are close to the theoretical values.Finally,11 actual commercially available samples,including five camel-milk and six camel-milk-powder samples were analyzed,which revealed that only camel signature peptides were detected in 10 samples,while camel and bovine signature peptides were both detected in one camel-milk-powder sample.The ingredient list of the latter sample revealed that it contained whole milk powder from an unidentified source;therefore,we infer that the bovine signature peptides originate from the whole milk powder.These signature peptides also demonstrate the necessity and practical significance of establishing this identification method.