Identification of bioactive peptides in a functional yogurt by micro liquid chromatography time-of-flight mass spectrometry assisted by retention time prediction

In this study we used micro liquid chromatography coupled to time-of-flight mass spectrometry (microLC–TOF-MS) for separation and identification of bioactive peptides in a yogurt marketed as an antihypertensive functional food. An appropriate sample clean-up using solid-phase extraction (SPE) allowed detection of a large number of low-molecular-mass bioactive peptides by reversed-phase microLC–TOF-MS. The preliminary identification was solely based on the experimental monoisotopic molecular mass values (Mexp). Later, we evaluated the correlations between predicted normalized elution time (NET) and experimental normalized retention times (t′rt′r) values to describe the retention behavior of the proposed sequences. The assistance of retention prediction proved to be useful to improve reliability of the identification, avoiding misinterpretations and solving some identity conflicts. After revision, the identity of only fifty bioactive peptides was confirmed. Significant number of these peptides was reported as angiotensin converting enzyme (ACE) inhibitors and nine of them were antihypertensive. The presence of peptide sequences with other biological activities such as antibacterial, antithrombotic, antioxidant, cell modulation, immune or phagocytosis stimulation, epitopes of B cells and opioid agonists was also confirmed.

► We developed a method for analysis of small molecules in yogurt by microLC–TOF-MS.
► We identified bioactive peptides.
► We refined identification using retention prediction.