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 predigestion of milk proteins and the consequent release of antibacterial peptides may provide a selective advantage through evolution by protecting both the mother's mammary gland and her nursing offspring from infection.

Figure 1

Example extracted compound chromatograms from identified peptides. The numbers indicate the position in the protein sequence the peptides were derived from. P₁ indicates the peptide has 1 phosphorylation. Polyimmunoglobulin receptor, PIGR; β-casein, B-CN; butyrophilin, BTN.

Figure 2

Example tandem mass spectrum of peptide RETIESLSSEESITEYK from B-CN identified by both X!Tandem and MS-GFDB.

Figure 3

Number of unique peptides identified by protein of origin. PIGR: polymeric immunoglobulin receptor; BSAL: Bile salt-activated lipase; COHA1: collagen α-1(XVII) chain; MRC1: Macrophage mannose receptor 1; PRB1P: Proteins represented by one peptide.

Figure 4

Unique peptides found from all 5 mothers aligned against parent protein sequence for butyrophilin, osteopontin, mucin-1 and polymeric immunoglobulin receptor.

Figure 5

Antibacterial activity of human milk peptides incubated with 10⁵ E. coli. Wells were loaded as follows: B2, 6 μg/μL milk peptides; B4, 0.6 μg/μL milk peptides; C3, 0.06 μg/μL milk peptides; C5, 0.006 μg/μL milk peptides; F1, 1 μg/μL maganin; F4, 1 μg/μL human defensin-6; D6, nanopure water.

Figure 6

Antibacterial activity of human milk peptides incubated with 10⁶ S. aureus. Wells were loaded as follows: C5, 8 μg/μL milk peptides; D2, 4 μg/μL milk peptides; F2, 1 μg/μL maganin; F4, 1 μg/μL human defensin-6; E3, nanopure water.