Human Milk Proteins and Their Glycosylation Exhibit Quantitative Dynamic Variations during Lactation

Abstract

Background: Proteins in human milk are essential and known to support the growth, development, protection, and health of the newborn. These proteins are highly modified by glycans that are currently being recognized as vital to protein structure, stability, function, and health of the intestinal mucosa. Although milk proteins have been studied, the quantitative changes in milk proteins and their respective site-specific glycosylation are unknown.

Objective: This study expanded the analytical tools for milk proteins and their site-specific glycosylation and applied these tools to a large cohort to determine changes in individual protein concentrations and their site-specific N-glycosylation across lactation.

Design: A tandem mass spectrometry method was applied to 231 breast-milk samples from 33 mothers in Davis, California, obtained during 7 different periods of lactation. Dynamic changes in the absolute abundances of milk proteins, as well as variation in site-specific N-glycosylation of individual proteins, were quantified.

Results: α-Lactalbumin, β-casein, k-casein, and α-antitrypsin were significantly increased from colostrum to transitional milk (4.37 ± 1.33 g/L to 6.41 ± 0.72 g/L, 2.25 ± 0.86 g/L to 2.59 ± 0.78 g/L, 1.33 ± 0.44 g/L to 1.60 ± 0.39 g/L, and 0.09 ± 0.10 g/L to 0.11 ± 0.04 g/L, respectively; P < 0.002). α-Lactalbumin (37%), β-casein (9%), and lysozyme (159%) were higher in mature milk than in colostrum. Glycans exhibited different behavior. Fucosylated glycans of lactoferrin and high-mannose, undecorated, fucosylated, sialylated, and combined fucosylated + sialylated glycans of secretory immunoglobulin A increased during lactation even when the concentrations of the parent proteins decreased.

Conclusions: Proteins in healthy mothers vary dynamically through lactation to support the development of infants. Individual milk proteins carried unique glycan modifications that varied systematically in structure even with site specificity. The role of glycosylation in human milk proteins will be important in understanding the functional components of human milk. This trial was registered at clinicaltrials.gov as NCT01817127.

Keywords: fucosylation; human milk proteins; lactation; mass spectrometry; sialylation; site-specific N-glycosylation.

Copyright © American Society for Nutrition 2019.

Figures

FIGURE 1

MRM chromatograms of (A) peptides and (B) glycopeptides in human milk. Blue squares, green circles, yellow circles, red triangles, and purple diamonds represent N-acetylglucosamine, mannose, galactose, fucose, and sialic acid, respectively. AT, α-antitrypsin; J, J chain; α-Lact, α-lactalbumin; LF, lactoferrin; Lyso, lysozyme; MRM, multiple reaction monitoring; Oste, osteopontin; SC, secretory component.

FIGURE 2

Dynamics of human milk proteins across lactation in the order of decreasing concentration: (A) first 6 abundant proteins; (B) next 5 abundant proteins. Values are mean ± SDs, n = 33. AT, α-antitrypsin; α-Lact, α-lactalbumin; LF, lactoferrin; Lyso, lysozyme; Oste, osteopontin.

FIGURE 3

Percentage changes in protein concentrations across lactation (colostrum to week 24) for 11 proteins. Data are reported as mean values, n = 33.

FIGURE 4

Variations in site-specific glycosylation across lactation of selected glycoproteins: (A) lactoferrin, (B) heavy chain of IgA, (C) secretory component of IgA, (D) J chain, (E) IgG, (F) IgM, and (G) α-antitrypsin. Blue squares, green circles, yellow circles, red triangles, and purple diamonds represent N-acetylglucosamine, mannose, galactose, fucose, and sialic acid, respectively. Data are reported as log value (mean) of fold change relative to colostrum, n = 33. AT, α-antitrypsin; J, J chain; LF, lactoferrin; SC, secretory component.