Sequence determination of αs1-casein isoforms from donkey by mass spectrometric methods

Four co-eluting components, with experimentally measured Mr of 23 658, 23 786, 24 278 and 24 406 Da, were detected by reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) analysis in the dephosphorylated casein fraction of a milk sample collected at middle lactation stage from an individual donkey belonging to the Ragusano breed. By coupling RP-HPLC, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), enzymatic digestions, MALDI-TOFMS and capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analyses, the four components were identified as donkey’s αs1-CNs and their sequences completely characterized, using the known mare’s αs1-CN (GenBank Acc. No. AAK83668; Mr 23750.7 Da) as reference. The proteins with Mr of 23 786 and 23 658 Da differ in the presence of a glutamine residue at position 83 in the full-length component and present the amino acid substitutions Q8 →H and H115 →Y with respect to the mare’s αs1-CN. The other two components with Mr 24 406 and 24 278 Da, which also differ in the presence of a glutamine residue at position 88 in the full-length component, show the insertion of the pentapeptide HTPRE between Leu33 and the Glu34. The two αs1-CNs bearing the pentapeptide insertion were named variants A (202 amino acids; Mr 24 406) and A1 (201 amino acids; Mr 24 278), whereas the two αs1-CNs without the pentapeptide were named variants B (197 amino acids; Mr 23 786) and B1 (196 amino acids;Mr 23 658)