Xu Tao, Chen Jingyao, Yang Kai, Qiao Weicang, Zhao Junying, Chen Lijun
School of Bioengineering, Dalian Polytechnic University, Dalian, China.
National Engineering Research Center of Dairy Health for Maternal and Child, Beijing Sanyuan Foods Co. Ltd, Beijing, China.
Front Chem. 2022 May 10;10:872251. doi: 10.3389/fchem.2022.872251. eCollection 2022.
This study was aimed to establish a method for quantitatively determining the ratio of whey protein in the total protein of infant formula by respectively selecting two characteristic peptides from whey protein and casein and calculating the ratio between the characteristic peptides. A nanoliter high-performance liquid chromatography tandem high-resolution mass spectrometry (Q Exactive) was used to simultaneously detect the characteristic peptides of two main whey proteins and two main caseins. The characteristic peptides were calculated, predicted, and screened using the ExPASy website, and peptide information was confirmed by database retrieval after the analysis by using a high-resolution mass spectrometer. The matrix effect was compensated by comparing the characteristic peptides in whey protein with those in casein protein, in which isotope internal standards were not required. The influence of the changes of the protein content in whey protein and casein on the detection method was eliminated by the calculation formula designed by ourselves. In this detection method, the sample was stable in the total protein concentration range of between 0.1 and 0.4 mg/ml. In the simulated industrial processing environment, with desalted whey powder, the recovery rate was 98.63-113.33% under different spiked levels with good reproducibility (RSD<8%). The RSDs of intraday and interday precisions were 2.03-9.35% and 0.61-11.02%, respectively. The different processing procedures of samples had no significant impact on the detection of whey protein (RSD% for milk samples treated by different processing techniques was 2.97%). The quantitation method of whey protein was applied to evaluate the whey protein content in different brands of commercially available milk powder. In summary, the proposed method was applicable for quantitative analysis of whey proteins in the infant formula.
本研究旨在建立一种通过分别从乳清蛋白和酪蛋白中选择两种特征肽并计算特征肽之间的比例来定量测定婴儿配方奶粉总蛋白中乳清蛋白比例的方法。采用纳升高效液相色谱串联高分辨率质谱仪（Q Exactive）同时检测两种主要乳清蛋白和两种主要酪蛋白的特征肽。利用ExPASy网站对特征肽进行计算、预测和筛选，并在使用高分辨率质谱仪分析后通过数据库检索确认肽信息。通过比较乳清蛋白中的特征肽与酪蛋白中的特征肽来补偿基质效应，其中不需要同位素内标。通过我们自己设计的计算公式消除了乳清蛋白和酪蛋白中蛋白质含量变化对检测方法的影响。在这种检测方法中，样品在总蛋白浓度范围为0.1至0.4mg/ml时稳定。在模拟工业加工环境中，对于脱盐乳清粉，在不同加标水平下回收率为98.63 - 113.33%，重现性良好（RSD<8%）。日内和日间精密度的RSD分别为2.03 - 9.35%和0.61 - 11.02%。样品的不同加工程序对乳清蛋白的检测没有显著影响（不同加工技术处理的牛奶样品的RSD%为2.97%）。将乳清蛋白的定量方法应用于评估市售不同品牌奶粉中的乳清蛋白含量。综上所述，所提出的方法适用于婴儿配方奶粉中乳清蛋白的定量分析。
