Discovery and quantification of non-human proteins in human milk

The question whether and which non-human 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 non-human peptides/proteins in the complex milk sample makes them a challenging target to detect. Here, by deep proteome profiling we detected several non-human peptides, which could be grouped as non-human 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 by using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by reduction, alkylation and in-gel digestion. The resulted peptide mixtures were subjected to proteomics analysis. We were able to detect 1577 human proteins in human milk. Additionally, we identified 109 non-human peptides, of which 71 were grouped in to 36 non-human proteins. In the next step, we targeted 37 non-human peptides and 9 of them could be repeatedly quantified in human milk samples. Peptides/proteins originating from bovine milk products were the dominant non-human proteins observed, notably bovine caseins (α-S1-, α-S2-, β-, κ-caseins) and β-lactoglobulin.