The question whether and which nonhuman
peptides or proteins are
present in human milk was raised many decades ago. However, due to
cross-reactivity or nonspecific antibody recognition, the accuracy
of detection by immunochemical methods has been a concern. Additionally,
the relative low-abundance of nonhuman peptides/proteins in the complex
milk sample makes them a challenging target to detect. Here, by deep
proteome profiling, we detected several nonhuman peptides, which could
be grouped as nonhuman proteins. We next estimated their concentration
in human milk by combining data-dependent shotgun proteomics and parallel
reaction monitoring. First, we fractionated human milk at the protein
level and were able to detect 1577 human proteins. Additionally, we
identified 109 nonhuman peptides, of which 71 were grouped into 36
nonhuman proteins. In the next step, we targeted 37 nonhuman peptides
and nine of them could be repeatedly quantified in human milk samples.
Peptides/proteins originating from bovine milk products were the dominant
nonhuman proteins observed, notably bovine caseins (α-S1-, α-S2-,
β-, κ-caseins) and β-lactoglobulin. The method we
present here can be expanded to investigate more about nonhuman peptides
and proteins in human milk and give a better understanding of how
human milk plays a role in allergy prevention.