Improving Fractionation of Human Milk Proteins through Calcium Phosphate Coprecipitation 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 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.