Identification and characterization of a new beta-casein variant in goat milk by high-performance liquid chromatography with electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry

A new variant of beta-casein was detected in the casein fraction obtained from milk of a goat belonging to an autochthonous breed of southern Italy, "Argentata dell'Etna". Reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) analysis indicated that the new beta-casein variant, here named D, has a M(r) 15 Da higher than that of variant C previously described. The modification in the amino acid sequence responsible for the 15 Da difference in M(r) between variants C and D was determined by coupling trypsin digestion with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and RP-HPLC/ESI-MS, and it was demonstrated that it is due to the point mutation Val(207) --> Asn(207). The phosphorylation pattern of the new variant D was shown to be identical to that of variant C, as the protein shows two phosphorylation levels, 5 and 6P, occurring with comparable relative abundances. Ser35 was determined as one of the phosphorylation sites, whereas the others were probably analogous to those determined previously for the beta-Cn variant C, at Thr12 and Ser1517-19. The results reported here indicate that the combined use of RP-HPLC/ESI-MS, MALDI-TOFMS and MS/MS represents a powerful tool for the detection and characterization of minor components present in complex protein mixtures.