Separation and identification of β-casein from chinese human milk by ion exchange chromatography-matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry

Abstract

The selective precipitation of whole casein from skimmed milk was achieved by the addition of calcium salt under acidic pH. The effects of pH, centrifugal force and final concentration of CaCl2 on the separation of casein were studied by measuring the purity of final products using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that casein with the highest purity could be obtained with the pH of 4. 3, the centrifugal force of 10 400 g and the final concentration of CaCl2 of 60 mmol/L. The casein was processed with DEAE anion exchange chromatography and three peaks were obtained. Then the third peak (peak II) was identified with Western-Blot method and matrix-assisted laser desorption/ ionization time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). The identification of Western-Blot showed that peak III can combine with the specificity of human milk β-casein antibody, and it is proved to be human milk β-casein. The fingerprints of peak M were analyzed by Mascot searching, and the sequence coverage was 50%, further supporting it is human milk β-casein. In conclusion, an effective method to obtain human milk β-casein from milk samples through DEAE anion exchange chromatography was established, and it is suitable for the proteomics research requirements of the β-casein from human milk.