Key Laboratory of Groundwater Resources and Environment of the Ministry of Education, Jilin University, Changchun 130012, China.
Division of Chemical Metrology & Analytical Science, National Institute of Metrology, Beijing 100029, China.
Molecules. 2023 Jul 4;28(13):5200. doi: 10.3390/molecules28135200.
β-casein, a protein in milk and dairy products, has two main variant forms termed as A1 and A2. A1 β-casein may have adverse effects on humans. The fact that there is only one amino acid variation at the 67th position between A1 and A2 β-casein makes it difficult to distinguish between them. In this study, a novel method using characteristic thermolytic peptides is developed for the determination of A1 and A2 β-casein in milk. Firstly, caseins extracted from milk samples are thermolytic digested at 60 °C without any denaturing reagents required for unfolding proteins, which simplifies the sample pretreatment procedure. The characteristic thermolytic peptides (i.e., fragments 66-76 and 59-76 for A1 and A2 β-casein, respectively) selected to specifically distinguish A1 and A2 β-casein only have eleven or eighteen amino acid moieties. Compared with tryptic characteristic peptides with a length of 49 amino acid moieties, these shorter thermolytic characteristic peptides are more suitable for LC-MS analysis. This novel method, with the advantages of high specificity, high sensitivity, and high efficiency, was successfully applied for the analysis of six milk samples collected from a local supermarket. After further investigation, it is found that this method would contribute to the development of A2 dairy products for a company and the quality inspection of A2 dairy products for a government.
β-酪蛋白是牛奶和奶制品中的一种蛋白质，有两种主要的变体形式，分别称为 A1 和 A2。A1β-酪蛋白可能对人体有不良影响。A1 和 A2β-酪蛋白之间只有第 67 位氨基酸的一个变异，这使得它们很难区分。在这项研究中，开发了一种使用特征热解肽的新方法，用于测定牛奶中的 A1 和 A2β-酪蛋白。首先，从牛奶样品中提取的酪蛋白在 60°C 下进行热解消化，无需任何用于展开蛋白质的变性试剂，从而简化了样品预处理程序。选择用于特异性区分 A1 和 A2β-酪蛋白的特征热解肽（即 A1 和 A2β-酪蛋白的 66-76 和 59-76 片段）分别只有 11 或 18 个氨基酸残基。与 49 个氨基酸残基长的胰蛋白酶特征肽相比，这些较短的热解特征肽更适合 LC-MS 分析。该新方法具有特异性高、灵敏度高和效率高的优点，成功应用于当地超市采集的 6 个牛奶样品的分析。进一步调查发现，该方法将有助于一家公司开发 A2 乳制品和政府对 A2 乳制品的质量检查。
