Improving fractionation of human milk proteins through calcium phosphate co-precipitation and their rapid characterization by capillary electrophoresis

This work describes a simple sample pretreatment method for the fractionation of human milk proteins (into their two main groups: whey and caseins) prior to their analysis. The protein extraction protocol is based on the addition of calcium phosphate to non-adjusted pH human milk. The combination of calcium ions with phosphate results in an effective co-precipitation of caseins. To assess the suitability of this fractionation protocol, the protein extracts were analyzed by SDS-PAGE, LC-MS/MS and CE analysis. The results evidence a significant decrease in contamination of casein fraction with whey proteins and vice versa compared with the conventional isoelectric precipitation of caseins. In addition, CE fraction collection coupled to LC-MS/MS (off-line coupling) has been successfully applied to the identification of minor proteins in this complex matrix. The methodology presented here constitutes a promising tool to enlarge the knowledge of human milk proteome.