Discovery and Quantification of Nonhuman Proteins in Human Milk

Abstract

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.

Maternal Allergy and the Presence of Nonhuman Proteinaceous Molecules in Human Milk

“…This database is provided in the Supplementary Materials, the fasta database. Allergens were added to the database because of their immunological relevance and bovine milk proteins because the majority of the nonhuman proteinaceous molecules in human milk was previously shown to originate from bovine milk [2]. The selection of human and bovine milk proteins was made based on previous data analysis of human and bovine milk protein samples (data not published) using databases with all human or bovine proteins available in UniProtKB (both downloaded from UniProt on 16-10-2018).…”

“…Besides a vast number of human proteins and peptides, it also includes nonhuman intact proteins, large protein fragments, and peptides (later referred to as proteinaceous molecules). The presence of such molecules in human milk has been demonstrated decades ago with immunochemical analysis [1] and has recently been confirmed with mass spectrometry [2].…”

“…The identified peptide sequences (n = 14) correspond to a sequence coverage of (22%). In contrast to studies from other groups that removed these peptides from their data sets because of the use of BSA as quality control in their studies [2,4], we did not remove these peptides from our results. No evidence was found for carryover of BSA peptides in the LC system.…”

“…In a later study, this was extended with peptides from α-lactalbumin (ALA), κ-casein, β-casein, and lactoferrin [4]. Evidence for the presence of intact bovine caseins and BLG has recently been provided [2,4,5]. In addition to the bovine proteins and peptides, Zhu et al also identified several peptide sequences originating from other nonhuman species, which may include allergens [2].…”

“…Evidence for the presence of intact bovine caseins and BLG has recently been provided [2,4,5]. In addition to the bovine proteins and peptides, Zhu et al also identified several peptide sequences originating from other nonhuman species, which may include allergens [2]. So far, only peanut allergen has been identified with high sequence coverage by liquid chromatography tandem mass spectrometry (LC-MS/MS) [6].…”