Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy.
Visualisation of multiple isoforms of kappa-casein on 2-D gels is restricted by the abundant alpha- and beta-caseins that not only limit gel loading but also migrate to similar regions as the more acidic kappa-casein isoforms. To overcome this problem, we took advantage of the absence of cysteine residues in alpha(S1)- and beta-casein by devising an affinity enrichment procedure based on reversible biotinylation of cysteine residues. Affinity capture of cysteine-containing proteins on avidin allowed the removal of the vast majority of alpha(S1)- and beta-casein, and on subsequent 2-D gel analysis 16 gel spots were identified as kappa-casein by PMF. Further analysis of the C-terminal tryptic peptide along with structural predictions based on mobility on the 2-D gel allowed us to assign identities to each spot in terms of genetic variant (A or B), phosphorylation status (1, 2 or 3) and glycosylation status (from 0 to 6). Eight isoforms of the A and B variants with the same PTMs were observed. When the casein fraction of milk from a single cow, homozygous for the B variant of kappa-casein, was used as the starting material, 17 isoforms from 13 gel spots were characterised. Analysis of isoforms of low abundance proved challenging due to the low amount of material that could be extracted from the gels as well as the lability of the PTMs during MS analysis. However, we were able to identify a previously unrecognised site, T(166), that could be phosphorylated or glycosylated. Despite many decades of analysis of milk proteins, the reasons for this high level of heterogeneity are still not clear.
Holland
John W
JW
Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia. j.holland@imb.uq.edu.au
Deeth
Hilton C
HC
Alewood
Paul F
PF
D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D002364:Caseins / Q000032:analysis* / Q000235:genetics; D002417:Cattle; D003545:Cysteine / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D014644:Genetic Variation; D006031:Glycosylation; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002364:Caseins; D020033:Protein Isoforms; D003545:Cysteine