| 19862798 |
Cunsolo V, Cairone E, Fontanini D, Criscione A, Muccilli V, Saletti R, Foti S: Sequence determination of alphas1-casein isoforms from donkey by mass spectrometric methods. J Mass Spectrom. 2009 Dec;44(12):1742-53.
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 alpha (s1)-CNs and their sequences completely characterized, using the known mare's alpha (s1)-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 alpha (s1)-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 alpha (s1)-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 alpha (s1)-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). |
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