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SUMMARYThe primary structures of ovine α>s1-casein variants A, C and D (formerly called Welsh variant) were determined. Separation of variants from whole casein was achieved using a fast and reliable reversed-phase HPLC method. Extended structural characterization of the purified proteins using electrospray mass spectrometry, automated Edman degradation and peptide mapping by means of HPLC-fast atom bombardment-mass spectrometry demonstrated that the mature protein was a mixture of two molecular species that differed in the deletion of residues 141–148 and were therefore 199 and 191 residues long respectively. The 199 residue peptide chain, which accounted for ∼ 80% of the entire translated αsl-casein, was as long as its caprine and bovine counterparts, and had a 98 and 89 % degree of identity with those two proteins respectively. Nine serine residues (positions 12, 44, 46, 64 to 68 and 75) were fully phosphorylated in αsl-casein A, whereas Ser115and Ser41were phosphorylated by ∼ 50 and ∼ 20% respectively. The differences between the three genetic variants A, C and D were simple silent substitutions, which however involved the degree to which the protein was phosphorylated. Variant C differed from variant A in the substitution Ser13→> Pro13which determined the loss of the phosphate group on site 12 of the protein chain, SerP12→>Ser12. A further substitution, SerP68→> Asn68caused the disappearance of both phosphate groups in the phosphorylated residues Ser64and Ser66in variant D; in this last casein variant there was no evidence of phosphorylation at Ser41.

SUMMARYThe heterogeneity of ovine β-casein, apparent after discontinuous gel electrophoresis at alkaline pH and gel isoelectric focusing either by staining with Coomassie blue or immunoblotting, was investigated in detail. Electrospray mass spectrometry was used to characterize ovine casein by direct examination of whole casein samples. β-Casein in individual milks consisted of several components which were characterized through the accurate determination of their molecular masses. It was demonstrated that protein species occurring simultaneously in individual ovine milks differed mainly in the number of phosphate groups.

SummaryThe non-protein nitrogen (NPN) of samples of Parmigiano-Reggiano cheese ripened for 6 and 15 months was fractionated by ion-exchange chromatography on a Cu2+-Chelex column to separate oligopeptides from free amino acids. Peptide components were isolated by reversed-phase HPLC and identified by fast atom bombardment–mass spectrometry (FAB–MS). Only the NPN fraction of 6 month old cheese samples contained enough peptides to be further characterized. On the basis of FAB–MS spectral results, 39 oligopeptides were identified, the main components being phosphopeptides. Two sets of both intact and partly dephosphorylated peptides, accounting for a total of 19 phosphopeptides, were formed by the hydrolysis of β–casein and belonged to regions 1–20 and 6–28 of β–casein. The formation and potential role of these peptides in cheese is discussed.

SummaryThe isolation and identification of low molecular mass peptides formed during the ripening of Parmigiano-Reggiano cheese is described. A strategy was used based on the fractionation of nitrogenous material using chemical methods followed by HPLC to isolate peptides and fast atom bombardment-mass spectrometry to identify them. It was found that the majority of cheese oligopeptides arose from the proteolysis of β-casein. Several phosphopeptides and oligopeptides known in vivo to be biologically active have also been identified during the ripening of cheese.