IDENTIFICATION OF A NEW GENETIC VARIANT OF BOVINE BETA-CASEIN USING REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND MASS-SPECTROMETRIC ANALYSIS

Various components of the beta-casein fraction from bovine milk were s eparated by preparative reversed-phase high-performance liquid chromat ography (RP-HPLC). They included the genetic variants beta A(1), beta A(2), beta A(3), and an unknown component previously denoted beta X [S . Visser et al., J. Chromatogr. 548 (1991) 361-370]. Tryptic digests o f these components were compared by RP-HPLC and most peaks were analys ed by mass spectrometry (MS). The tryptic map of beta X was closest to that of beta A(1), but with a few mutually different peak components. Electrospray ionisation MS revealed that in the beta X map these comp onents had relative molecular masses of 16 higher than the correspondi ng ones in the beta A(1) map. The main differential peaks represented the 114-169 fragments of beta A(1) and beta X, respectively, which wer e both purified and then cleaved with cyanogen bromide. In the resulti ng mixtures, each of which contained three fragments, the correspondin g peptides representing the 145-156 sequence showed the 16 relative mo lecular mass difference. In beta X this sequence contained a Leu resid ue at position 152 instead of the Pro-152 in beta A(1), as established by fast-atom bombardment MS-MS. The Leu could be discriminated from a n Ile residue by the presence of a side-chain-specific, D-type fragmen t ion in the MS-MS spectrum of the beta X CNBr peptide. The sequence o f the two homologous 145-156 fragments was confirmed by regular amino acid sequence analysis. In accordance with internationally accepted gu idelines for the nomenclature of milk proteins, the new genetic varian t has been named beta-casein F-5P.