Identification and characterization of a new β ‐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 β ‐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 β ‐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 β ‐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. Copyright © 2004 John Wiley & Sons, Ltd.