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

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 spectrometry had strong anti-interference ability,high sensitivity,high accuracy and good reproducibility.It is expected that the comparability of β-LG quantitative results between different laboratories will be improved.In addition,the combination of new ultrasensitive mass spectrometry with stable-isotope labelling techniques has advanced to a point that some studies with radioactive isotopes can now be readily replaced by stable-isotope techniques.This trend is expected to continue and this isotope method will be a key component of the design of new clinical studies currently under way.At the same time,with the widespread use of stable isotope markers in food allergy,metabolomics,proteomics,clinical pharmacology and other fields,the demand for stable isotope labeled compounds is expected to further increase.