Smolenski, Grant A.Kelton, WilliamTock, Adriënne2026-02-022026-02-022025https://hdl.handle.net/10289/17908Dipeptidyl peptidase 4 (DPP4) is a serine exopeptidase that cleaves dipeptides from the N-terminus, preferably when proline occupies the penultimate position. This enzyme plays a key role in maintaining glucose homeostasis by degrading insulin secreting incretins, such as gastric inhibitory peptide and glucagon-like peptide 1, thereby reducing insulin secretion. Consequently, inhibiting DPP4 activity has become a major strategy for mitigating the effects of Type 2 diabetes mellitus. A functional food approach involves inhibiting this enzyme with DPP4-inhibiting peptides that are generated from dairy products. Milk and colostrum are associated with various health benefits, largely due to the presence of many bioactive peptides. A significant portion of these are proline-rich peptides (PRPs), some of which exhibit DPP4 inhibitory properties. While many DPP4-inhibitory dairy peptides have been identified or predicted through ‘in silico’ analysis, the variations across different species and between milk and colostrum have yet to be explored. This research identified new PRPs in cow, goat, sheep, and deer milk and colostrum and demonstrated that some of these peptides have DPP4-inhibitory activity. Peptide identification and characterisation were performed using liquid chromatography tandem mass spectrometry, a new resin-based DPP4-binding procedure, and in vitro DPP4 inhibition assays. PRPs represented 20-30% of the identified peptides within each sample, with sheep colostrum reaching 35%. Peptides from deer had the lowest alignment with the Cervidae proteome, likely due to database incompleteness. Exact peptide sequence overlap among all samples was minimal. Peptides were chosen for DPP4 inhibition assays based on the resin pull-down assay results, presence of proline in the penultimate position, abundance, length, and origin. Out of the 21 selected peptides, 14 showed over 50% inhibition at 250 µM. Interestingly, these peptides revealed that greater proline content did not reliably predict DPP4 inhibition, which is contradictory to reports in the literature. The top six DPP4-inhibiting peptides were selected for IC50 determination, with values ranging from 46.7 to 103.2 µM. Even though these peptides were all less potent than the reference peptide inhibitor Diprotin A (IC50 of 2.69 ± 1.09 µM, n = 6), they all showed high abundance of at least 105. Additionally, most peptides represented novel sequences that were not previously reported in the literature as DPP4-inhibiting peptides. Pooled peptide fractions from each sample did not show inhibition at the tested concentration, although permeate from a dairy plant showed 34.41% ± 8.27 (n = 3) DPP4 inhibition at only 1.5% w/w of the permeate concentration. AlphaFold 3 predictions revealed that these peptides and Diprotin A docked at similar positions within the DPP4 active site. These findings provide the basis for future studies on interspecies differences, application of novel binding assays to find DPP4 inhibitors, and further exploration of dairy peptides as potential DPP4 inhibitors.enAll items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.Thesis