Identification of bioactive peptides in hypoallergenic infant milk formulas by capillary electrophoresis–mass spectrometry

In this study, we use capillary electrophoresis–mass spectrometry (CE–MS) for the identification of bioactive peptides in hypoallergenic infant milk formulas (IF), which are complex bovine milk protein hydrolysates. A sample clean-up pretreatment with a citrate buffer containing dithiothreitol and urea followed by solid-phase extraction (SPE) with different reversed-phase commercial cartridges was investigated to achieve optimum detection sensitivity in CE–MS. SPE with C18, StrataX and Oasis HLB cartridges allowed detection of the largest number of low molecular mass components, but combination of C18 and StrataX results was enough to achieve an excellent coverage of the studied IF. The monoisotopic molecular mass values of the low molecular mass components obtained by capillary electrophoresis ion-trap mass spectrometry (CE–IT–MS) allowed the tentative identification of nine bioactive sequences. Only the identification of five of them could be confirmed when accurate mass measurements were performed by capillary electrophoresis time-of-flight mass spectrometry (CE–TOF–MS), namely LKP, IPY, ALPM, PGPIHN and VAGTWY, which were reported to present angiotensin-converting enzyme (ACE) inhibitory and antimicrobial activity (only VAGTWY).