Andres Guerrero; Stephanie Contreras; Dave Dallas; Lauren Wu; Jennifer Smilowitz; Daniela Barile; Bruce German; Carlito Lebrilla
University of California, Davis, Davis, CA
Peptidomics analysis of milk yields bioactive peptides that vary during lactation and disease.
Human milk, as other biofluids, contains proteolytic enzymes that produce endogenous peptides. A library of endogenous peptides found in human milk with post-translational modifications was created through the use of nano-LC and tandem mass spectrometry. This library was used to develop a method to identify peptides rapidly allowing monitoring with quantitation nearly 1000 peptides with nano-LC MS. The method was used to examine changes in the peptide abundances during lactation. A comprehensive understanding of the endogenous proteolytic activity in human milk can be achieved to compare different milk samples. In addition because many of the peptides have antimicrobial activity, we examined the effect of peptide production and abundances during diseases such as mastitis infection.
Peptide separation was performed using 25 ?L of human milk that was spiked with an internal standard. The samples were defatted and trichloroacetic acid was added to precipitate the peptides. The samples were cleaned up by solid phase extraction using a C-18 column. The samples were analyzed on a nano-LC Q-TOF with a C-18 chip to obtain MS and MS/MS data. The tandem mass spectrometry information from each sample was exported as an MGF file and uploaded to X! tandem to identify peptides present in each milk sample analyzed. When the peptides in milk were identified an in-house software was used to visualize the most abundant peptides present within a protein.
The peptide milk library contains peptide fragments from different proteins along with their masses, retention times, and post-translational modifications that may occur on certain amino acids that include phosphorylation, oxidation, deamination, and water loss. The peptide milk library was used to identify peptides from different proteins of mothers with and without mastitis. Based on the analysis, we found that the vast majority of the peptides came from five proteins in milk: ?-casein, ?-casein, polymeric immunoglobulin, osteopontin, and butryophilin. By using an in-house software we were able to assign the peptide fragments within these proteins and identify locations where the abundances varied the most. This software also allowed us to locate the peptides within these proteins that had phosphorylation sites. For example, with ?-casein the peptides that had 1, 2, and 3 phosphorylation sites were all located at the N-terminus of the protein, whereas the peptides with no phosphorylation sites where found throughout the protein. Analysis of milk from mothers with mastitis were performed. The total peptide abundances from five proteins were summed over several time points to determine whether peptide abundances varied during mastitis. There was a general trend of a decrease in the peptide abundances around the time the mother had mastitis.