Andres - Copy
Andres Guerrero1; Dave C. Dallas2; Nora Khaldi3; Daniela Barile2; J. Bruce German2; Carlito B. Lebrilla1
1UC Davis, Chemistry Department, Davis, CA; 2UC Davis, Food Science Department, Davis, CA; 3Food for Health Ireland, University College Dublin, Belfield, Ireland
NOVEL ASPECT: A comprehensive MS method was used to analyze endogenous peptides from human milk that were shown to be antimicrobial.
A novel, streamlined and high-throughput analytical approach has been developed to capture and identify a comprehensive set of peptides produced by the in vivo proteolytic digestion of human milk.
By employing an iterative exclusion list on the tandem MS analysis and multiple injections, the number of unique endogenous peptides identified increased by nearly 5-fold compared to a single tandem identification run. An annotated library of endogenous peptides sequences and their PTMs has been constructed to streamline yield a method for the rapid and comprehensive analysis of peptides in milk.
The results indicate that the digestion of proteins in the mammary yield specific peptide sequences from distinct proteins that exert protective influence on both the infant and the mother.
Milk peptide purification was performed through centrifugation, protein precipitation with TCA and C18 solid phase extraction.
Peptide samples were analyzed in the positive mode on an Agilent nanoLC-Q-TOF with a C18 chromatographic chip. Automated precursor selection based on ion abundance was employed for tandem fragmentation. After each analysis, newly identified peptides were added to an exclusion list for subsequent repeat experiments. Molecular ions on the exclusion list were ignored by the instrument and hence were not fragmented again. This approach allowed deeper exploration of the sample.
Peptide identification was accomplished using two searchers, MS-GFDB and X!Tandem against a milk protein library (1472 entries) based on a query to the Uniprot database. Peptide results were accepted with p-values lower than 0.01.
Extensive peptidomic analysis of milk allowed the identification of 508 unique peptide sequences from 27 different proteins including β-casein (62% of the total), polymeric immunoglobulin receptor, butyrophilin, αs1-casein, osteopontin, κ-casein and mucin-1. However, neither the number of peptides found nor their relative signal intensities appears to follow the natural abundances of their respective protein origin. For example, highly abundant proteins in milk such as α-lactalbumin and lactoferrin are not represented.
Peptides identified ranged in length from 6 to 37 amino acids. 163 peptides (32% of the total) were phosphorylated. By comparison with the database Phosphosite it was determined than 35 of these sequences had a previously unknown phosphorylation site.
Interestingly, the peptides identified were not randomly distributed across the overall protein sequence but were focused on specific sites for butyrophilin, κ-casein, osteopontin, mucin-1, perilipin and polymeric immunoglobulin receptor. β-casein is unique in this regard as almost the entire sequence was represented.  These results indicate that proteolysis in the mammary gland is selective—released peptides were drawn only from specific proteins and typically from selected parts of the parent sequence.
Selective hydrolysis suggests specific functional roles for the endogenous peptides. A large number (62) of the endogenous peptides were found to share at least 57% of their sequence with known functional peptides,based on a comparison with functional peptide databases (BIOPEP, PeptideDB, CAMP and APD2). Fifty-five of the peptides matched known antibacterial sequences, while seven peptides matched immunomodulatory sequences.
The antimicrobial activity of the milk peptides was tested in a radial diffusion assay against a Gram-negative (E. coli) and a Gram-positive (S. aureus) species.  We show for the first time that the endogenous peptides in milk are antimicrobial. S. aureus is a bacteria commonly found in mastitis suggesting that the peptides not only protect the infant but the mother as well.