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

Abstract

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.

Genetic Characterisation ofCSN2Gene inGirgentanaGoat Breed

“…At least, ten alleles have been identified in goat CSN2 gene. In particular, seven of these alleles (A, A1, C, C1, E, 0, and 0') were characterised at DNA level (Rando et al, 1996;Persuy et al, 1999;Chessa et al, 2005Chessa et al, , 2008Cosenza et al, 2005), whereas B and D alleles were described only at protein level (Mahé and Grosclaude, 1993;Galliano et al, 2004). Another variant has been found by Chianese et al (2007) at protein level but it was not yet characterised.…”

“…Another variant has been found by Chianese et al (2007) at protein level but it was not yet characterised. Furthermore, the genetic variants A, A1, B, C, C1, D, and E are associated with a normal -casein content in milk (about 5 g/L per allele) (Roberts et al, 1992;Mahé and Grosclaude, 1993;Neveu et al, 2002;Galliano et al, 2004;Cosenza et al, 2005;Caroli et al, 2006) while the two null alleles (0 and 0'), are associated with a non-detectable amount of this protein (Ramunno et al, 1995;Persuy et al, 1999).…”

Effects on Production Traits of Haplotypes Among Casein Genes in Norwegian Goats and Evidence for a Site of Preferential Recombination

“…At least five variants of CSN2 are described, including a null allele associated with no protein expression (Mahe and Grosclaude 1993;Galliano et al 2004). Likewise at least five different alleles have been described for CSN1S2 (Recio et al 1997;Martin et al 1999;Erhardt et al 2002).…”

Genetic Factors that Regulate Milk Protein and Lipid Composition in Goats

“…Cosenza et al (2007) have also reported a single nucleotide polymorphism at the CSN2 promoter but this variant has not been named yet. With regard to the A, B, C, D and E variants, they have been identified by means of proteomic techniques such as isoelectric focusing (Mahé and Grosclaude 1993), peptide mass fingerprinting and tandem mass spectrometry (Neveu et al 2002), reversed phase HPLC/electrospray ionization mass spectrophotometry (Galliano et al 2004) and immunoelectrophoretic analysis (Chianese et al 2007). Molecular characterization of the null variants revealed that the 0 allele contains a 1 bp deletion in the 5'end of exon 7 that induces the appearance of a premature stop codon resulting in a 20-fold reduction in mRNA synthesis and the generation of a much shorter protein (72 vs 223 amino acids, Persuy et al 1999).…”