Analysis of disulphide linkages in bovine κ-casein oligomers using two-dimensional electrophoresis

• Published in: Electrophoresis 2008-06, Vol.29 (11), p.2402-2410
• Main Authors: Holland, John W, Deeth, Hilton C, Alewood, Paul F
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Caseins - chemistry; Cattle; Disulfides - analysis; Disulphide bond; Disulphide-linked peptide; Electrophoresis, Gel, Two-Dimensional - methods; Mass spectrometry; Milk - chemistry; Milk protein; Molecular Sequence Data; Protein Isoforms - isolation & purification; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry; κ-Casein
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.200700840

Disulphide bonds play an important role in protein structure and function. Bovine κ-casein (κ-csn), an important glycoprotein in milk, contains two cysteines that can form disulphide bonds. On 2-D gels run under nonreducing conditions the κ-csn in milk presented a complex pattern of monomers and disulphide-linked oligomers. Trains of spots corresponding to monomers to hexamers were observed as a result of the participation of different glycoforms and phosphoforms in oligomer formation. The dimers and trimers ran as doublets on the gel and analysis of the disulphide-linked peptides released from them after in-gel tryptic digestion showed they were the result of different disulphide linkages. The linkages were confirmed by MSMS. When milks with electrophoretically distinct genetic variants of κ-csn were mixed and run on 2-D gels, they retained their distinct patterns indicating that disulphide exchange reactions or disulphide 'scrambling' was not occurring during 2-D analysis. The patterns observed represent the native distribution of κ-csn in milk at harvest. The role and significance of the disulphide bonding of κ-csn are discussed.