Improving Fractionation of Human Milk Proteins through Calcium Phosphate Coprecipitation and Their Rapid Characterization by Capillary Electrophoresis

Abstract

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 nonadjusted pH human milk. The combination of calcium ions with phosphate results in an effective coprecipitation of caseins. To assess the suitability of this fractionation protocol, the protein extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), LC-MS/MS, and capillary electrophoresis (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.

Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (mid‐2018–2022)

“…A review of proteomic analysis of milk and dairy products embracing also the applied analytical methods was recently published [198]. CE-UV and CE off-line coupled with LC-MS/MS were utilized for the analysis of human milk proteins [199]. CE was found to be an appropriate method for the analysis of milk proteins and the assessment of the authenticity of dairy products [200][201][202].…”