Analysis of major bovine milk proteins by on-line high-performance liquid chromatography and electrospray ionization-mass spectrometry

Abstract

Summary - Proteins from skim milk, paracaseinate and whey have been successfully analyzed by reverse-phase high-pertormance liquid chromatography (RP-HPLC) cou pied with electrospray ionization mass spectrometry (ESI-MS). The major milk proteins were identified by comparison of molecular masses determined by ESI-MS to molecular masses calculated from amino acid composition deduced from primary structures and cDNA sequences for some proteins. This method has permilted the simultaneous identification of caseins and whey protein variants. Observed molecular masses of major milk proteins were found to be 19038.0 ± 2.2 Da forK-CN A-l P (number of experiments n =6); 19007.0 ± 1.1 Da for K-CN B-1P (n = 4); 25230.0 ± 2.1 Da for as2-CN A (n = 9); 23617.2 ± 1.3 Da for as1-CN B-8P (n = 14); 24 092.0 ± 1.7 Da for ~-CN B-5P (n = 6); 24 025.3 ± 1.0 Da for ~-CN A 1-5P (n =14); 23 984.8 ± 0.7 Da for ~-CN A2_5P (n =14); 18278.3 ± 2.2 Da for the monomeric form of ~-LG B (n = 5); 18 364.8 ± 1.6 Da for the monomeric form of ~-LG A (n = 5); 14 179.21 ± 3.14 Da for a-LA (n =5). Hence an accuracy of 0.01 % was obtained by ESI-MS analysis. It was also shown that the on-line coupling of HPLC with ESI-MS offers a very promising alternative for studying the proteolysis and determining the specificity of used enzymes in some technological treatments as shown for milk-clotting enzymes.