β-酪蛋白,牛奶和乳制品中的蛋白质,有两种主要的变体形式称为A1和A2。A1β-酪蛋白可能对人类有不利影响。A1和A2β-酪蛋白之间在第67位只有一个氨基酸变异的事实使得难以区分它们。在这项研究中,开发了一种使用特征性热解肽测定牛奶中A1和A2β-酪蛋白的新方法。首先,从牛奶样品中提取的酪蛋白在60°C下进行热解消化,而无需任何解折叠蛋白质所需的变性试剂,这简化了样品预处理程序。特征性的热解肽(即,A1和A2β-酪蛋白的片段66-76和59-76,分别)选择以具体区分A1和A2β-酪蛋白仅具有11个或18个氨基酸部分。与长度为49个氨基酸部分的胰蛋白酶特征肽相比,这些较短的热解特征肽更适合于LC-MS分析。这种新颖的方法,具有高特异性的优点,高灵敏度,效率高,已成功用于分析从当地超市收集的六个牛奶样品。经过进一步调查,发现该方法将有助于公司开发A2乳制品和政府对A2乳制品的质量检查。
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.