Detection of intact bovine milk proteins after simulated gastrointestinal infant digestion using UHPLC − HRMS

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.