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.

An aliquot of 300 µl thawed whole milk sample was centrifuged at 1000 × g for 1 h at 4°C to separate the upper-fat layer and lower skimmed milk. For PRM analysis, several non-human peptides were selected. For the selected peptides, stable isotope-labeled standards terminated with C-terminal heavy Arg/Lys were purchased. The heavy labeled peptides were mixed equally together, and a PRTC mixture was spiked in to monitor and calibrate the scheduled RT window. Mixed peptide digestion, heavy labeled peptides and PRTC mixture were analyzed by LC-MS/MS, as described above for shotgun proteomics, except that the LC separation used a 35min gradient of 13-40% buffer B and the mass analyzer was a Q-Exactive High Field X quadrupole-Orbitrap mass spectrometer. Detection was done with a resolution of 60,000 using an AGC setting of 2e5, maximum IT of 128 ms, 1 microscan, 1.4 m/z isolation window, 27% normalized collision energy and 5 min retention time window. Skyline was used to build a spectral library and assign integrated fragment peak.Skyline was used to build a spectral library and assign integrated fragment peak. Summing extracted peak areas of top 5 (or fewer) ranked non-interfered fragment ions per each precursor ion were used for calculating ratio of light to heavy in the pair of non-human peptide and its stable isotope standard. Non-human peptide amount was generated by the ratio of light to heavy multiplying amount of stable isotope-labeled peptide.

Human milk samples (n=6) were obtained from milk collections forming part of the MediDiet study from healthy Italian mothers aged between 25 and 40 in a period between 5 and 7 weeks postpartum. All samples were collected from women delivering at term childbirth. Samples were stored at -80°C until analysis. This trial was registered in the Dutch Trial Register (www.trialregister.nl; NTR3468).