David Dallas1; Andres Guerrero1; Nora Khaldi2; Bruce German1; Daniela Barile1; Mark Underwood1; Carlito Lebrilla1
1University of California, Davis, Davis, CA; 2UC Dublin, Dublin, Ireland
NOVEL ASPECT: This study represents a novel application of high-throughput peptidomics to investigate milk protein digestion in term and premature infants.
Decades of research demonstrate that human milk protein digestion releases peptide fragments with beneficial antimicrobial and immunomodulatory functions for the infant. However, as previous studies were based on in vitro digestion of milk, the relevance of these findings is unknown. For the first time, we analyze peptides released from human milk in the infant stomach in vivo. Released peptide sequences are determined via our novel, high-throughput peptidomics technique and potential functions are examined. Because premature infants have lower acid and enzyme production in the stomach than term infants, we hypothesize that premature infants do not produce the same functional milk peptides as term infants. This study compares digestion between these infant groups to identify missing functional peptides in premature infants.
In the UC Davis Medical School’s neonatal intensive care unit, gastric samples were obtained from term and premature infants after 2 hours of digestion. Infants already intubated for medical conditions preventing normal feeding, but without digestive disorders were selected. Peptides were extracted from all samples in 96-well plate format by centrifugal delipidation, acid precipitation of proteins, and C18 solid phase extraction clean-up.Peptides were measured by nano-LC-Q-TOF with automated peak selection for tandem fragmentation. Tandem spectra were searched in X!Tandem with no enzyme selected.
A library of identified peptides with exact mass and retention time was applied back to all samples for peak volume extraction. Univariate statistical testing was employed for peptide abundance comparison between term and premature infants.

Analysis of peptides with the LC-MS/MS technique revealed hundreds of unique peptides in all mother’s milk and infant gastric samples. The number of unique peptides increased 3-fold from intact milk to gastric samples, demonstrating that digestion does occur in the infant stomach. Specific proteins that were not digested in the mammary gland, like lactoferrin and alpha-lactalbumin, are degraded within the infant stomach of both term and premature infants. Evidence for digestion in the stomach is surprising because conditions in the infant stomach are far from optimal for the only known infant gastric protease:
pepsin. Enzyme analysis of the cleavage sites revealed that pepsin is active at low levels in the infant stomach as early as the first week of life. Moreover, this analysis revealed that milk proteases that were initially blocked by antiproteases in the mammary gland become functional in the infant stomach and are responsible for the majority of cleavages. This demonstrates that milk continues to guide its own digestion within the infant gut. The factor causing activation of these milk enzymes in the infant stomach remains unknown.Statistical analysis of the differences in gastric digestion between term and premature infants is underway. Identification of milk enzymes as the main contributors to gastric digestion demonstrate that differences in proteases and antiproteases between term and premature mother’s milk may contribute to differences in peptide release in the stomach.

Approximately 10% of the identified peptides were homologous with known antimicrobial or immunomodulatory peptides. Differences in these functional peptides may contribute to the variance in health outcomes between term and premature infants.