MS-Based Characterization of αs2-casein isoforms in donkey’s milk

The primary structure of four as2-casein (CN) isoforms, present as minor components in the dephosphorylated CN fraction of amilk 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 as2-CN (GenBank Acc. No. CAV00691; Mr 26 028 Da) as reference. Proteins, withexperimentally measured Mr of 25 429, 21 939, 25 203 and 21 713 Da, were isolated by the combined use of reversed-phasehigh-performance liquid chromatography (RP-HPLC) and two-dimensional polyacrylamide gel electrophoresis. The major spotof each gel, corresponding to a single protein, was digested by trypsin, a-chymotrypsin and endoproteinase Glu-C. The resultingpeptide mixtures were analyzed by matrix-assisted laser desorption/ionization time-of-flightmass spectrometry and capillaryRP-HPLC/nano-electrospray ionization tandem mass spectrometry, and the data obtained were used for the sequencedetermination. The isoforms are produced from differential splicing events involving exons 4, 5 and 6 and parts of the exon 17.