MS-based characterization of α _(s2)-casein isoforms in donkey's milk

摘要

The primary structure of four α _(s2)-casein (CN) isoforms, present as minor components in the dephosphorylated CN fraction of a milk sample collected in Eastern Sicily from an individual donkey belonging to the Ragusano breed at middle lactation stage, was determined, using the known donkey's α _(s2)-CN (GenBank Acc. No. CAV00691; M _r 26 028 Da) as reference. Proteins, with experimentally measured M _r of 25 429, 21 939, 25 203 and 21 713 Da, were isolated by the combined use of reversed-phase high-performance liquid chromatography (RP-HPLC) and two-dimensional polyacrylamide gel electrophoresis. The major spot of each gel, corresponding to a single protein, was digested by trypsin, α-chymotrypsin and endoproteinase Glu-C. The resulting peptide mixtures were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary RP-HPLCano-electrospray ionization tandem mass spectrometry, and the data obtained were used for the sequence determination. The isoforms are produced from differential splicing events involving exons 4, 5 and 6 and parts of the exon 17.