Heterogeneity of the bovine kappa-casein caseinomacropeptide, resolved by liquid chromatography on-line with electrospray ionization mass spectrometry.
Microheterogeneity occurs in the population of caseinomacropeptides (residues 106-169 of kappa-casein) due to variation in the extent and type of oligosaccharide linked to this phosphoglycopeptide. Although caseinomacropeptide A variant (CMPA) was poorly resolved using reversed-phase high-performance liquid chromatography (RP-HPLC) with spectrophotometric detection, it could be analysed with on-line electrospray-ionization mass spectrometry (ESI-MS). From the already established O-linked glycan chains at least fourteen glycosylated forms of CMPA were identified, besides the non-glycosylated and multiphosphorylated (1, 2 or 3 phosphate groups) peptides, giving a maximum number of eighteen known forms. Major subcomponents in CMPA are disialylated species. A maximum of three out of the five potential glycosylation sites were found to be substituted with carbohydrate chains in the most highly glycosylated forms, which may contain up to six N-acetylneuraminic acid residues per molecule. A minor form, diphosphorylated with one disialylated chain, was also detected. From these results, it was shown that the on-line coupling of HPLC with ESI-MS offers a very promising alternative for the analysis of complex mixtures.
Mollé
D
D
Institut National de la Recherche Agronomique, Laboratoire de Recherches de Technologie Laitière, Rennes, France.
Léonil
J
J
D000818:Animals; D002240:Carbohydrate Sequence; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
D002364:Caseins; D010446:Peptide Fragments; C065989:caseinomacropeptide