高效液相色谱-同位素稀释质谱法准确定量牛乳中β-乳球蛋白  

Accurate Quantification of β-Lactoglobulin in Bovine Milk by HPLC-IDMS

作　　者：陈燕秋 徐增益 喻静兰 李良君 徐仲杰 雷雯 CHEN Yan-qiu;XU Zeng-yi;YU Jing-lan;LI Liang-jun;XU Zhong-jie;LEI Wen(Shanghai Stable Isotope Detection and Application R&D Professional Technical Service Platform,National Isotope Engineering Technology Research Center,Shanghai Research Institute of Chemical Industry Co,Ltd,Shanghai 200062,China;Chengdu Institute of Food Inspection,Chengdu 611130,China)

机构地区：[1]上海化工研究院有限公司,国家同位素工程技术研究中心上海分中心,上海市稳定同位素检测与应用研发专业技术服务平台,上海200062 [2]成都市食品检验研究院,四川成都611130

出　　处：《质谱学报》2023年第3期436-441,I0005,共7页Journal of Chinese Mass Spectrometry Society

摘　　要：β-乳球蛋白(β-LG)是牛乳的主要致敏蛋白之一,亟需开发一种准确且可追溯的准确定量分析方法。本研究利用胰蛋白酶酶解β-LG后,采用同位素稀释质谱(IDMS)法对特定多肽进行定量分析。同时考察氘标记特征多肽IDAL*NENK(D_(6)-Leu)及碳氮双标记特征多肽IDAL*NENK(^(13)C_(6),^(15)N-Leu)作为内标对色谱行为和检测结果的影响,并进行方法学验证。结果表明,本方法的回收率为90.1%~102.7%,变异系数(CVs)<8.0%。分析来自国内市场的5个样本,CVs<6.5%,合成成本较低的氘标记多肽在蛋白质定量中可作为^(13)C、^(15)N标记多肽的可靠替代。本方法抗干扰能力强、灵敏度高、准确度高、重现性好,有助于提高β-LG定量结果在不同实验室之间的可比性。Milk is one of the most common and widespread allergenic foods.Milk allergy is an adverse immunological reaction to milk proteins of different mammalian species.β-Lactoglobulin(β-LG) is one of the main allergenic proteins of cow's milk.There is an urgent need to develop an accurate and traceable method to accurately quantify β-LG.Based on the known β-lactoglobulin sequence,the Skyline tool was used to simulate the β-lactoglobulin digestion process.After digestion of β-LG with trypsin,the tryptic peptides in the samples were detected selectively by MS/MS.In short,the peptides were searched with MS full scan of respective digested milk protein.Three characteristic peptides of β-lactoglobulin were screened by primary local sequence search tool(basic local alignment search tool,BLAST) and Uniprot database.Finally,one of the characteristic peptides with the highest response intensity and the best stability was selected for further verification by HPLC-MS/MS and quantitative studied by multiple reaction monitoring(MRM).In this work,the specific peptides were quantified by isotope dilution mass spectrometry(IDMS) after trypsin digestion of β-LG.At the same time,the effects of deuterium-labeled characteristic peptide IDAL*NENK(D_6-Leu) and carbon-nitrogen dual-labeled characteristic peptide IDAL*NENK(^(13)C_6,^(15)N-Leu) as internal standards on chromatographic behavior and detection results were investigated,and the methodology was verified.The method showed a good linear relationship within its own range.The limits of detection and limits of quantification were 0.001 9-0.002 2 g/L and 0.006 4-0.007 4 g/L,respectively.The recoveries ranged from 90.1% to 102.7%,and the coefficients of variation(CVs) were less than 8.0%.Analysis of 5 samples from the domestic market showed CVs were less than 6.5%.Deuterium-labeled peptides with lower synthetic cost could be used as reliable substitutes for ^(13)C and ^(15)N-labeled peptides in protein quantification.The established quantitative method of isotope dilution mass spec

关 键 词：液相色谱-串联质谱(LC-MS/MS) β-乳球蛋白(β-LG) 同位素稀释质谱(IDMS) 特征多肽 

分 类 号：O657.63[理学—分析化学]