Kristen Beck, Genome Center, University of California Davis, USA
Kristen Beck1, Darren Weber2, Brett Phinney2 , Jennifer Smilowitz3, Katie Hinde4, Bo Lönnerdal5, Ian Korf1, Danielle G. Lemay1§
1Genome Center, University of California Davis, USA
2Proteomics Core, University of California Davis, USA
3Department of Food Science and Technology, University of California Davis, USA
4Department of Human Evolutionary Biology, Harvard University, USA
5Department of Nutrition, University of California Davis, USA
Objective: Milk has been well established as the optimal nutrition source for infants, yet there is still much to be understood about its molecular composition in humans and non-human primates. Therefore, our objective was to develop and compare comprehensive milk proteomes for human and rhesus macaques, the most widely used non-human primate in biomedical research. This comparative biology approach highlights differences in early neonatal nutrition between the species.
Methods: Until this point, the high incidence of interfering macronutrients, pervasive post-translational modifications, and abundant casein and whey proteins have been substantial barriers to comprehensive milk proteomics. Existing methods require a large sample volume that has been prohibitive of utilizing milk from non-human primates where sample availability is more limited. Therefore we have developed a small volume proteomics technique to complete the first high throughput molecular comparison of human and rhesus macaque milk proteins.
Whole milk (15ul) is fractionated using gradient SDS-PAGE and gel slicing which effectively removes the interfering macronutrients and isolates high abundance casein and whey proteins into their respective gel fragment. In addition, one of the most predominant post-translational modifications of milk proteins is glycosylation. Therefore, we incubated gel slices with PNGase F to cleave the N-linked glycans exposing more of the peptide chain. In its first utility in milk proteomics, we found it to greatly increase protein detection during tandem mass spectrometry.
Results: Using the aforementioned novel proteomics technique, we identified 1,606 and 518 proteins in human and macaque milk, respectively (~5% protein FDR, < 1% peptide FDR). This is a considerable expansion on the total number of proteins previously identified in human and is the first comprehensive macaque milk proteome. Using a reciprocal best match BLASTp approach and clustering in-paralogs with InParanoid (v 4.1), we identified 396 orthologous protein clusters present in both human and macaque milk. Notably, there were 88 proteins with differential abundance between the two species (p-value < 0.05 after adjusting for multiple hypothesis testing). Nearly all differentially abundant proteins are higher in human milk relative to macaque. Some of the most differentially abundant proteins include lactoferrin, bile salt activated lipase, polymeric immunoglobulin receptor, alpha-1 antichymotrypsin, 4-trimethylaminobutyraldehyde dehydrogenase and clusterin. Notably, human proteins with 2-fold greater normalized weighted spectra, compared to macaque, are associated with the development of the gastrointestinal tract, the immune system, and the brain. Conclusions: Overall, we demonstrate the effectiveness of our small volume, high throughput proteomics method to reveal the most comprehensive human milk proteome to date and the first complete macaque milk proteome. The proteins observed are consistent with the perspective of ‘secondary altriciality’ that has been used to describe the human development stage at birth in which we are adapted to be less developed at birth as a derived feature from our primate relatives. Our human milk proteome analyses suggest that mother’s milk nurtures infant maturation through higher quantities of specific proteins associated with development of the gastrointestinal tract, the immune system, and the brain than observed for macaque. These findings imply that infant formula could be improved by supplementation with specific milk proteins.