Endogenous human milk peptide release is greater after preterm birth than term birth

Abstract

Background: Hundreds of naturally occurring milk peptides are present in term human milk. Preterm milk is produced before complete maturation of the mammary gland, which could change milk synthesis and secretion processes within the mammary gland, leading to differences in protein expression and enzymatic activity, thereby resulting in an altered peptide profile.

Objective: This study examined differences in peptides present between milk from women delivering at term and women delivering prematurely.

Methods: Nano-LC tandem mass spectrometry was employed to identify naturally occurring peptides and compare their abundances between term and preterm human milk samples at multiple time points over lactation. Term milk samples were collected from 8 mothers and preterm milk was collected from 14 mothers. The 28 preterm and 32 term human milk samples were divided into 4 groups based on day of collection (<14, 14-28, 29-41, and 42-58 d).

Results: Preterm milk peptide counts, ion abundance, and concentration were significantly higher in preterm milk than term milk. Bioinformatic analysis of the cleavage sites for peptides identified suggested that plasmin was more active in preterm milk than term milk and that cytosol aminopeptidase and carboxypeptidase B2 likely contribute to extensive milk protein breakdown. Many identified milk peptides in both term and preterm milk overlapped with known functional peptides, including antihypertensive, antimicrobial, and immunomodulatory peptides.

Conclusion: The high protein degradation by endogenous proteases in preterm milk might attenuate problems because of the preterm infant's immature digestive system. This trial was registered at clinicaltrials.gov as NCT01817127.

Keywords: carboxypeptidase B2; cytosol aminopeptidase; functional peptide; human milk; mass spectrometry; peptidomics; plasmin; premature; term.

FIGURE 1

Number, ion intensities, and concentrations of peptides in preterm and term human milk over lactation time. Number of peptides in preterm and term milk over lactation time (A). Intensities of peptides in preterm and term milk over lactation time (B). Concentrations of peptides in preterm and term human milk over lactation time (C). Values are means ± SEMs. Number of observations (n) in each group is shown in Table 1. Asterisks indicate significant differences between preterm and term milk: **P < 0.01, ***P < 0.001.

FIGURE 2

Number and intensity of peptides in preterm and term human milk by protein of origin. Number of peptides identified in preterm and term milk by protein of origin (A). Intensity of peptides in preterm and term milk by protein of origin (B). Values are means ± SEMs; n = 32 for term milk, n = 28 for preterm milk. Asterisks indicate significant differences between preterm and term milk: **P < 0.01, ***P < 0.001. CASA1, α_s1-casein; CASB, β-casein; OSTP, osteopontin; Other, combination of all other protein precursors identified; PIGR, polymeric immunoglobulin receptor.

FIGURE 3

Intensity of peptides in preterm and term human milk by protein of origin over lactation time. Values are means ± SEMs. Number of observations (n) in each group is shown in Table 1. Asterisks indicate significant differences between preterm and term milk: *P < 0.05, **P < 0.01, ***P < 0.001. CASA1, α_s1-casein; CASB, β-casein; OSTP, osteopontin; Other, combination of all other protein precursors identified; PIGR, polymeric immunoglobulin receptor.

FIGURE 4

Predicted enzyme activity of plasmin, carboxypeptidase B2, cytosol aminopeptidase, cathepsin D, and elastase in preterm and term human milk. Values are means ± SEMs; n = 32 for term milk, n = 28 for preterm milk. Asterisks indicate significant differences between preterm and term milk: *P < 0.05, ***P < 0.001.

FIGURE 5

Predicted enzyme activity of plasmin in preterm and term human milk over lactation period. Values are means ± SEMs. Number of observations (n) in each group is shown in Table 1. Asterisk indicates significant difference between preterm and term milk: *P < 0.05.

FIGURE 6

Predicted activity of enzymes responsible for the endogenous human milk protein degradation based on the sum of peptide intensities. Values are means ± SEMs; n = 60. CASA1, α_s1-casein; CASB, β-casein; OSTP, osteopontin; PIGR, polymeric immunoglobulin receptor.

FIGURE 7

Proteolytic map of α_s1-casein in preterm and term human milk samples determined by mapping peptide intensities onto the α_s1-casein protein sequence. Values are means ± SEMs; n = 32 for term milk, n = 28 for preterm milk.