Determination of the phosphorylation level and deamidation susceptibility of equine beta-casein.
beta-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine beta-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's beta-casein (25 514 +/- 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27-34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the beta-casein isolated from Haflinger mare's milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30. Moreover, the equine beta-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif (135)Asn-Gly(136). Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions.
Girardet
Jean-Michel
JM
Laboratoire des BioSciences de l'Aliment, UC INRA 885, Faculté des Sciences et Techniques, Université Henri Poincaré-Nancy 1, Vandoeuvre-lès-Nancy, France. Jean-Michel.Girardet@scbiol.uhp-nancy.fr
Miclo
Laurent
L
Florent
Sabrina
S
Mollé
Daniel
D
Gaillard
Jean-Luc
JL
D000469:Alkaline Phosphatase / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006736:Horses; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D013058:Mass Spectrometry; D008892:Milk / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D002364:Caseins; D010446:Peptide Fragments; D020033:Protein Isoforms; D000469:Alkaline Phosphatase; D014357:Trypsin