Extensive in vivo Human Milk Peptidomics Reveals Specific Proteolysis Yielding Protective Antimicrobial Peptides

Abstract

Milk is traditionally considered an ideal source of the basic elemental nutrients required by infants. More detailed examination is revealing that milk represents a more functional ensemble of components with benefits to both infants and mothers. A comprehensive peptidomics method was developed and used to analyze human milk yielding an extensive array of protein products present in the fluid. Over 300 milk peptides were identified originating from major and many minor protein components of milk. As expected, the majority of peptides derived from β-casein, however no peptide fragments from the major milk proteins lactoferrin, α-lactalbumin and secretory immunoglobulin A were identified. Proteolysis in the mammary gland is selective—released peptides were drawn only from specific proteins and typically from only select parts of the parent sequence. A large number of the peptides showed significant sequence overlap with peptides with known antimicrobial or immunomodulatory functions. Antibacterial assays showed the milk peptide mixtures inhibited the growth of Escherichia coli and Staphylococcus aureus. The pre-digestion of milk proteins and the consequent release antibacterial peptides may provide a selective advantage through evolution by protecting both the mother's mammary gland and her nursing offspring from infection.

Bioactive peptides released from in vitro digestion of human milk with or without pasteurization

“…Except residues 51-59, the presence of these peptides in undigested HM has been observed previously (14,15). Two peptides of residues 1-18 and 1-23 are considered human counterparts of caseinophosphopeptides because they have the "acid motif " SerP-SerP-SerP-Glu-Glu, essential for the mineral-carrier potential (11).…”

“…Other peptides were released after in vitro digestion and showed quite similar profiles in HM and pasteurized HM. Interestingly, there was no peptide identified which was released from residues 29-153, and Dallas et al also reported a similar observation (15). One possible explanation can be attributed to five glycosylation sites of OPN in human milk, all of which are located in residues 29-153 (36); glycosylated peptides are possibly released but lack of information regarding exact oligosaccharide structures makes them impossible to be identified by this peptidomic method.…”

“…Articles RESULTS Peptides in undigested HM were analyzed prior to the analysis of digesta as background data (Figure 1), because some of the protein-derived peptides are potentially present in undigested HM (14,15). As can be seen, a large number of peptides were observed.…”

“…The most abundant peptides are derived from β-CN, reflecting its high susceptibility to plasmin-mediated proteolysis as well as the high content of this casein subunit in HM (14,16). Focusing on bioactive peptides in undigested HM, as shown in Table 1 (14,15,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30), two caseinophosphopeptide homologues, a possible opioid peptide or propeptide, and an antibacterial peptide were identified.…”

Mechanistic Peptidomics: Factors That Dictate Specificity in the Formation of Endogenous Peptides in Human Milk

“…Only a few attempts have been reported to characterize the naturally occurring peptide content in human milk, but those have focused on the description of peptides produced from a small number of milk proteins (20,39). More recently, our group developed an analytical procedure to purify and analyze the endogenous peptide content in human milk (40).…”