ANALYSIS OF MAJOR BOVINE-MILK PROTEINS BY ONLINE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

Proteins from skim milk, paracaseinate and whey have been successfully analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) coupled 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 permitted the simultaneous identification of caseins and whey protein variants. Observed molecular masses of major milk proteins were found to be 19 038.0 +/- 2.2 Da for kappa-CN A-1P (number of experiments n = 6); 19 007.0 +/- 1.1 Da for kappa-CN B-1P (n = 4); 25 230.0 +/- 2.1 Da for alpha(s2)-CN A (n = 9); 23 617.2 +/- 1.3 Da for alpha(s1)-CN B-8P (n = 14); 24 092.0 +/- 1.7 Da for beta-CN B-5P (n = 6); 24 025.3 +/- 1.0 Da for beta-CN A(1)-5P (n = 14); 23 984.8 +/- 0.7 Da for beta-CN A(2)-5P (n = 14); 18 278.3 +/- 2.2 Da for the monomeric form of beta-LG B (n = 5); 18 364.8 +/- 1.6 Da for the monomeric form of beta-LG A (n = 5); 14 179.21 +/- 3.14 Da for alpha-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.