Identification of minor proteins of human colostrum and mature milk by two-dimensional electrophoresis

Two-dimensional electrophoresis (2-DE) followed by electroblotting and microsequencing is considered to be the most powerful method for the isolation and characterization of proteins. In this paper, we report the separation and determination of the N-terminal and/or internal amino acid sequences of the minor proteins of human colostral and mature milk by 2-DE and microsequencing. In order to analyze the minor proteins of human milk, we use immunoabsorbents to remove three major proteins, alpha-lactalbumin, lactoferrin and secretory immunoglobulin A. The major proteins removed by this process accounted for about 79 and 93 % of the total whey proteins of mature and colostral milk, respectively. The remaining milk proteins were then separated by isoelectric focusing gel electrophoresis between pH 3 and 10, and subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Approximately 400 spots were detected in both colostral and mature milk by silver staining after 2-DE. Twenty-two major, well-resolved proteins tout of 400) were microsequenced (N-termini as well as internal). These include fatty acid binding protein, beta(2)-microglobulin, complement C4, clusterin, alpha(1)-antritrypsin, lysozyme C, alpha- and beta-casein, prealbumin, serotransferrin, fructose-bisphosphate aldolase A, and beta-casein fragments. No major differences in the protein patterns were observed between the minor proteins of colostrum and mature milk, indicating that the minor proteins remained relatively constant during lactation. These results suggest that the minor milk proteins are important for the health and development of breast-fed infants throughout lactation.