Equine α_S1-casein: Characterization of alternative splicing isoforms and determination of phosphorylation levels

Hydrophobic interaction chromatography of Haflinger mare's sodium caseinate. The sample (100mg) was loaded onto a TSK Phenyl-5PW column (Interchim, Montluçon, France). A linear gradient from 480 to 37mM sodium phosphate, 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, quantities of 40μg of protein were loaded on the gel. Proteins were stained by Coomassie Brilliant Blue. eCN = equine sodium caseinate; the main bands were numbered from 1 to 14 according to Egito et al. (2002).

Two-dimensional PAGE of Haflinger mare's α_S1-casein isoforms contained in the hydrophobic interaction chromatography fractions before (F3 and κ-CN–free F4) and after (F3d and κ-CN–free F4d) double dephosphorylation by alkaline phosphatase and acid phosphatase. Quantities of 100μg of proteins were loaded in the immobilized pH gradient gel strips. MM = apparent molecular mass.

Reconstructed masses from liquid chromatography electrospray ionization mass spectrometry [Q/Star XL spectrometer, MDS Sciex, Toronto, Canada; molecular mass (M_r)±1] of Haflinger mare's α_S1-casein isoforms contained in the dephosphorylated fractions F3d and κ-CN–free F4d. α_S1-CN = full-length α_S1-casein; d = dephosphorylated; ΔX = deletion of the amino acid sequence encoded by exon X; ΔQ = deletion of the N-terminal residue (Gln) of the region encoded by exon 11; 0P = no phosphate group.

Exon modular structure of the Haflinger mare's full-length α_S1-casein deduced from mRNA sequence (GenBank accession number AY049939; Milenkovic et al., 2002). The exon nomenclature is that used by Miranda et al. (2004). The potential sites of phosphorylation are in bold characters. The exons that can be alternatively spliced are indicated in italics. The Gln residue that can be deleted is underlined.

Fractionation by ion-exchange chromatography of Haflinger mare's α_S1-casein previously prepared by hydrophobic interaction chromatography. The sample (30mg) was loaded onto a TSK DEAE-5PW column (Amersham Pharmacia, Uppsala, Sweden). A linear gradient from 0.08 to 0.8 M NaCl was applied for 70min at a flow rate of 0.5 mL/min. Composition of each subfraction (SF1 to SF6) was checked by urea-PAGE (inset). For that, quantities of 40μg of proteins were loaded in the gel. SF = subfraction; the main bands were numbered from 8 to 12 according to Egito et al. (2002).

Reconstructed masses from liquid chromatography electrospray ionization mass spectrometry [Q/Star XL spectrometer, MDS Sciex, Toronto, Canada; molecular mass (M_r)±1] of Haflinger mare's α_S1-casein subfractions (SF1 to SF6) obtained by ion-exchange chromatography. α_S1-CN = full-length α_S1-casein; ΔX = deletion of the region encoded by exon X; ΔQ = deletion of the N-terminal residue (Gln) of the amino acid sequence encoded by exon 11; P = phosphate group.