Identification of a new genetic variant of bovine ß-casein using reversed-phase high-performance liquid chromatography and mass spectrometric analysis

• Published in: Journal of Chromatography A Vol. 711; no. 1; pp. 141 - 150
• Main Authors: Visser, Servaas, Slangen, Charles J., Lagerwerf, Fija M., Van Dongen, William D., Haverkamp, Johan
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 08-09-1995
• ISSN: 0021-9673
• DOI: 10.1016/0021-9673(95)00058-U

Various components of β-casein fraction from bovine milk were separated by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). They included the genetic variants
ßA
1,
βA
2,
βA
3, and an unknown component previously denoted ßX [S. Visser et al., J. Chromatogr. 548 (1991) 361–370]. Tryptic digests of these components were compared by RP-HPLC and most peaks were analysed by mass spectrometry (MS). The tryptic map of ßX was closest to that of
ßA
1, but with a few mutually different peak components. Electrospray ionisation MS revealed that in the ßX map these components had relative molecular masses of 16 higher than the corresponding ones in the
ßA
1 map. The main differential peaks represented the 114–169 fragments of
ßA
1 and ßX, respectively, which were both purified and then cleaved with cyanogen bromide. In the resulting mixtures, each of which contained three fragments, the corresponding peptides representing the 145–156 sequence showed the 16 relative molecular mass difference. In ßX this sequence contained a Leu residue at position 152 instead of the Pro-152 in
ßA
1, as established by fast-atom bombardment MS-MS. The Leu could be discriminated from an Ile residue by the presence of a side-chain-specific, D-type fragment ion in the MS-MS spectrum of the ßX CNBr peptide. The sequence of the two homologous 145–156 fragments was confirmed by regular amino acid sequence analysis. In accordance with internationally accepted guidelines for the nomenclature of milk proteins, the new genetic variant has been named β-casein F-5P.