Sequence determination of a donkey’s CSN1S2 duplicate gene product by high resolution mass spectrometry

Within the frame of our research line aimed to the systematic investigation donkey’s milk protein composition,1-6 a casein component, with experimentally high resolution measured Mr of 16316.1748 Da, was detected by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) coupled on-line with an hybrid ESI-MS LTQ/Orbitrap mass spectrometry 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 high resolution top-down and bottom-up approaches, the unknown component was identified as a new isoform of donkey’s αs2-CN variant B, which should be encoded by the duplicate gene CSN1S2 II. The primary structure of this protein was completely characterized using as reference the m-RNA derived amino acid sequence of the donkey’s s2-CN variant B reported in GenBank (Acc. No. CAX65660.2; Mr 16721.27 Da). The MS data derived sequence revealed that the investigated protein differs from the GenBank reported sequence for: i) the amminoacid point substitution Ser131→Leu; ii) the insertion of the hexapeptide KLELTK between the Asp50 and Glu51; iii) the absence of the peptide E3IKHVSSSE11, which should be encoded by the exon 3; and iv) the absence of the phenylalanine encoded by exon 9. References 1 Saletti, R.; Muccilli, V.; Cunsolo, V.; Fontanini, D.; Capocchi, A.; Foti, S.; Journal of Mass Spectrometry, 2012, 47, 1150. 2 Cunsolo, V.; Muccilli, V.; Fasoli, E.; Saletti, R.; Righetti, P.G.; Foti, S.; Journal of Proteomics, 2011, 74, 2083. 3 Cunsolo, V.; Cairone, E.; Fontanini, D.; Criscione, A.; Muccilli, V.; Saletti, R.; Foti, S.; Journal of Mass Spectrometry, 2009, 44, 1742. 4 Cunsolo, V.; Cairone, E.; Muccilli, V.; Saletti, R.; Foti, S.; Rapid Communication in Mass Spectrometry, 2009, 23, 13, 1907. 5 Cunsolo, V.; Saletti, R.; Muccilli, V.; Foti S.; Journal of Mass Spectrometry, 2007, 42, 1162. 6 Cunsolo, V.; Costa, A.; Saletti, R.; Muccilli, V.; Foti, S.; Rapid Communication in Mass Spectrometry, 2007, 21, 1438.