Identification And Characterization Of A New Beta-Casein Variant In Goat Milk By High-Performance Liquid Chromatography/Electrospray Ionization-Mass Spectrometry And Matrix Assisted Laser Desorption/Ionization Mass Spectrometry

A new variant of b-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 b-casein variant, here named D, has a Mr 15 Da higher than that of variant C previously described. The modification in the amino acid sequence responsible for the 15 Da difference in Mr 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 Val207! Asn207. 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 b-Cn variant C, at Thr12 and Ser15,17–19. 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.