Abstract: After removing high-abundance proteins from breast milk, we applied proteomics to analyze low-abundance proteins with special important biofunctions in breast milk. Mature milk about 1 month old was used for this study, and high-abundance proteins were removed by immunoaffinity chromatography or ProteoMiner low-abundance protein enrichment kit, followed by proteomic analysis. The results showed that both methods were effective in removing high-abundance proteins. A total of 186 low-abundance proteins were identified by the two methods, 49 proteins of which were identified by immunoaffinity chromatography, and 172 by ProteoMiner low-abundance protein enrichment kit. According to bioinformatics analysis and gene ontology (GO) functional annotation analysis, the functions of the identified proteins mainly included serving as cellular components, participating in biological processes in cells and exerting binding activities. Using the Pathway database as a reference, these proteins were found to be involved in 201 metabolic pathway branches, including the PI3K-Akt signaling pathway and the phospholipase D signaling pathway. Compared with the Cluster of Orthologous Groups of proteins (COG) database, these proteins mainly participated in such biological activities as carbohydrate transport and metabolism, and signal transduction mechanisms. These results not only help to understand the functions of low-abundance proteins in breast milk, but also provide a theoretical basis for healthy growth and disease prevention in infants and young children as well as provide technical support for research on the protective mechanism of breastfeeding and on breast diseases.

Key words: breast milk; polyclonal antibody; immunoaffinity chromatography; proteome