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Description
The presence and relative abundance of intact protein after gastrointestinal digestion is an important aspect of the evaluation of digestion efficiency and the assessment of protein allergenicity. The detection of intact proteins after gastrointestinal digestion is commonly done using gel electrophoretic applications (SDS-PAGE). These are highly sensitive, however in some cases SDS-PAGE does not allow for an unambiguously identification of the food proteins due to 1.) overlap of the bands of the digestive enzymes with the proteins of interest and 2.) the overlap of larger protein break-down products with lower molecular weight proteins. Therefore, this project aimed to explore whether UHPLC-HRMS can be used as a fast, sensitive, and unambiguous tool to monitor intact protein after gastrointestinal digestion. This was done on the example of bovine milk proteins. Skimmed raw milk was applied to a static in vitro gastrointestinal model to simulate infant digestion [1]. Samples were taken after 60 min in the gastric phase (GP60) and after 10 min intestinal digestion (IP10) and measured on an UHPLC-HRMS system (timsTOF Pro 2, Bruker Daltonics, Billerica, Massachusetts, USA). Additionally, samples were applied to SDS-PAGE to compare the two methods. Detection of β-lactoglobulin variant A was possible in the soluble phase of the digests GP60 and IP10 using UHPLC HRMS, while caseins were not detectable. This was in line with the analysis by SDS PAGE. In contrast, the band of α-lactalbumin (ALA) was visible on SDS PAGE at both digestion times points however with UHPLC-HRMS it could only be detected in GP60 but not in IP10. This could indicate that the band for ALA that is visible on SDS-PAGE in IP10 corresponds to a partially hydrolysed ALA with a hydrolysis degree that is too low to resolve with SDS-PAGE. However, this requires further confirmation.
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