Sequence determination of alphas1-casein isoforms from donkey by mass spectrometric methods.
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).
Cunsolo
Vincenzo
V
Department of Chemical Science, University of Catania, Viale A. Doria 6, I-95125 Catania, Italy. vcunsolo@unict.it
Cairone
Elisa
E
Fontanini
Debora
D
Criscione
Andrea
A
Muccilli
Vera
V
Saletti
Rosaria
R
Foti
Salvatore
S
D000485:Allergens / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D018397:Equidae; D005260:Female; D013058:Mass Spectrometry / Q000379:methods*; D008892:Milk / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
D000485:Allergens; D002364:Caseins; D010446:Peptide Fragments