Abstract
Nanoflow-HPLC-tandem mass spectrometry (MS/MS) was used to analyze the peptide fraction of breast milk samples collected from a single non-atopic donor on different days (10 samples) after receiving an oral load of cow's milk (by drinking 200 mL of bovine milk). In addition, breast milk was sampled from the same lactating mother over a 6-h period at five time points after drinking cow's milk. We aimed to trace the intra-individual variability and to define a time profile of the excretion of dietary peptides into breast milk. Overall, 21 peptides exclusively originating from both bovine caseins and whey proteins with no match within the human milk proteome were identified in the breast milk samples. These peptides were missing in the breast milk obtained from the mother after a prolonged milk- and dairy-free diet (three samples). The time course of cow's milk-derived β-Lg f(125–135) and β-casein f(81–92) in breast milk was determined from the MS ion intensity of the peptide signals. No intact cow's milk gene products were detected by HPLC-MS/MS analysis and Western blotting with anti-β-Lg antibody, but dot-blot analysis confirmed the occurrence of β-Lg fragments in the enriched peptide fraction of breast milk. These data suggest shifting the analytical perspective for the detection of dietary food allergens in breast milk from intact proteins to digested peptide fragments. The possible sensitization and elicitation potential or the tolerogenic properties of such low amounts of dietary peptides for the breastfed newborns remain to be explored.
Highlights
Breast milk contains antigenically active food allergens arising from the mother’s diet [1]
Due to an intense activity of endogenous proteases, human milk is intrinsically rich in oligopeptides [29], which interfere with the mass spectrometry (MS)-based detection of trace amounts of possible foreign peptides arising from the mother’s diet
More than 1,200 peptides derived from endogenous proteins were identified by HPLC-MS/MS in the 12% TCAsoluble peptide fraction of each breast milk sample, as already reported in our previous work [24]
Summary
Breast milk contains antigenically active food allergens arising from the mother’s diet [1]. This assertion is mainly based on immunochemical data and clinical investigations carried out in the 1980s and 90s [4,5,6]. In those years, it had been clearly established that food allergens derived from a mother’s diet can elicit clinical manifestations of adverse reactions in exclusively breastfed susceptible newborns, with an incidence rate of 0.5% [7,8,9,10]. Food allergens in human milk have been reported to vary over a very wide range of concentrations from just above 0.1 to >1,000 ng/mL [3], but low ppb (ng/mL) levels are probably the most realistic figures [14].
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