Two-dimensional cartography of equine β-casein variants achieved by isolation of phosphorylation isoforms and control of the deamidation phenomenon

Exon modular structure of the equine β-casein determined by Miranda et al. (2004). The putative phosphorylation sites are indicated with an asterisk according to Miclo et al. (2007). The nonenzymatic deamidation site identified by Girardet et al. (2006) is double underlined. Other hypothetical deamidation sites, which would undergo peptide bond cleavage, are single underlined. The exons that can be alternatively spliced are framed with a dotted line. The splicing sites involved in the low molecular mass (M_r) β-casein generation are indicated by arrows. Exon 9, which would be only translated in the case of exon 8 splicing, is indicated in italics (translated according to cDNA structure determined by Lenasi et al., 2003).

The mechanism of Asn deamidation reaction at neutral and basic pH (according to Xie and Schowen, 1999). The α-amino of the residue C-flanking to the Asn makes a nucleophilic attack on the Asn side chain to form a succinimide intermediate. The cyclic imide undergoes hydrolytic attack at both carbonyl centers, leading to the iso-Asp and Asp products in a ratio of 2:1 to 4:1.

Hydrophobic interaction chromatography of Haflinger mare's sodium caseinate. The sample (100mg) was loaded onto a TSK Phenyl 5-PW column (Interchim, Montluçon, France). In the presence of 2.5 M urea, a linear gradient from 480 to 37mM PBS buffer, pH 6.4, was applied for 47min at a flow rate of 6 mL/min followed by an isocratic step at 37mM sodium phosphate for 12min. Composition of each collected fraction (F1 to F4) was checked by urea-PAGE (inset). For that, 40-μg quantities of proteins were loaded in gel. Proteins were stained by Coomassie Brilliant Blue. The presence of κ-casein in gel was specifically detected using Schiff's reagent after periodic acid oxidation. eCN = equine sodium caseinate.

Fractionation by ion-exchange chromatography of Haflinger mare's β-casein phosphorylation isoforms. The sample (F2, 15mg) was loaded onto a TSK DEAE 5-PW column (Interchim, Montluçon, France). A linear gradient from 0.08 to 0.8 M NaCl in 20mM imidazole buffer, pH 8.0, containing 3.3 M urea, was applied for 70min at a flow rate of 0.5 mL/min. Composition of each collected subfraction (F2.1 to F2.5) was checked by urea-PAGE (in inset). For that, 40-μg quantities of proteins were loaded onto gel. Proteins were stained by Coomassie Brilliant Blue. XP = number of phosphate groups.

Reconstructed masses from liquid chromatography electrospray ionization mass spectrometry of Haflinger's mare β-casein isoforms contained in subfractions F2.2 to F2.5. β-CN = full-length β-casein; Δ^X = deletion of the region encoded by exon X; XP = number of phosphate groups; cps = count per second.

Urea-PAGE monitoring of deamidation process of equine β-casein 5P and 6P isoforms isolated in the F2.3 and F2.4 subfractions, respectively. The reaction was carried out by incubation at 37°C in 150mM PBS buffer, pH 8.4, containing 0.02% (wt/vol) sodium azide and 10% (vol/vol) protease inhibitor cocktail for 24 and 48h. Fraction F2.5 containing native β-casein 7P was loaded onto the gel for migration comparison. P = phosphate group; XP = number of phosphate groups; d = deamidated; 0, 24, and 48 = time of incubation (h).

Two-dimensional electrophoresis (a) of the whole equine β-casein recovered in the F2 fraction by hydrophobic interaction chromatography and of its F2.2 to F2.5 ion-exchange chromatography subfractions, (b) of deamidated F2.3 and F2.4 (named F2.3d and F2.4d) subfractions. See text for deamidation reaction conditions. Quantities of 50μg of protein were loaded in the immobilized pH gradient strips. Proteins were stained by Coomassie Brilliant Blue. XP = number of phosphate groups; Δ^X = deletion of the region encoded by exon X; d = deamidated; M = molecular mass standards. The small arrow indicates the spot of β-CN 5P on each 2-dimensional profile by superposing the profile of F2.3. The dashed arrows show that the 6P and 5P/d spots and the 7P and 6P/d spots have the same positions on the 2-dimensional gel.

Two-dimensional electrophoresis of each of the F2.2 to F2.5 ion exchange chromatography subfractions mixed in a 1:1 mass ratio with the whole β-casein recovered in the F2 fraction by hydrophobic interaction chromatography. Quantities of 50μg of total protein were loaded in immobilized pH gradient strips. Proteins were stained by Coomassie Brilliant Blue. XP = number of phosphate groups, ΔX = deletion of the region encoded by exon X. Putative identification of the 3P variants is shown in italics.