Abstract
Four co‐eluting components, with experimentally measured M~r~ 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‐of‐flight 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‐TOF MS and capillary RP‐HPLC/nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) analyses, the four components were identified as donkey's α~__s__1~‐CNs and their sequences completely characterized, using the known mare's α~__s__1~‐CN (GenBank Acc. No. AAK83668; M~r~ 23750.7 Da) as reference. The proteins with M~r~ 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 Q^8^→H and H^115^→Y with respect to the mare's α~__s__1~‐CN. The other two components with M~r~ 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 Leu^33^ and the Glu^34^. The two α~__s__1~‐CNs bearing the pentapeptide insertion were named variants A (202 amino acids; M~r~ 24 406) and A~1~ (201 amino acids; M~r~ 24 278), whereas the two α~__s__1~‐CNs without the pentapeptide were named variants B (197 amino acids; M~r~ 23 786) and B~1~ (196 amino acids; M~r~ 23 658). Copyright © 2009 John Wiley & Sons, Ltd.