This study demonstrates the development and application of an ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC−HRMS) method for the rapid and sensitive identification of intact bovine milk proteins following simulated gastrointestinal infant digestion. The new method enables the differentiation between partially hydrolysed/modified and fully intact proteins. In the raw milk, intact α-lactalbumin was visible on SDS − PAGE until the end of the gastrointestinal digestion, while it was not detected with UHPLC−HRMS. Analysis of both raw and heated milk samples revealed that the method is applicable to various milk types. Interestingly, heated milk showed additional signals in the mass spectrum, indicating non-enzymatic post-translational modifications. The relative abundance of these proteoforms could be followed along digestion. These findings highlight the versatility and sensitivity of UHPLC−HRMS in elucidating protein structures and modifications, providing valuable insights into how simulated digestion affects milk protein composition.

中文翻译：

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使用 UHPLC − HRMS 检测模拟胃肠道婴儿消化后的完整牛奶蛋白


本研究展示了超高效液相色谱-高分辨率质谱 （UHPLC-HRMS） 方法的开发和应用，用于在模拟胃肠道婴儿消化后快速、灵敏地鉴定完整牛奶蛋白。新方法可以区分部分水解/修饰的蛋白质和完全完整的蛋白质。在生牛奶中，完整的 α-乳清蛋白在 SDS − PAGE 上可见，直到胃肠道消化结束，而用 UHPLC-HRMS 未检测到。对原料奶和加热奶样品的分析表明，该方法适用于各种牛奶类型。有趣的是，加热的牛奶在质谱中显示出额外的信号，表明非酶促的翻译后修饰。这些蛋白形式的相对丰度可以随着消化而进行跟踪。这些发现突出了 UHPLC-HRMS 在阐明蛋白质结构和修饰方面的多功能性和灵敏度，为模拟消化如何影响牛奶蛋白质组成提供了有价值的见解。