A signature protein-based method to distinguish Mediterranean water buffalo and foreign breed milk

► Internally deleted water buffalo αs1-casein variant is a signature protein. ► Primary structure of never signaled αs1-CN variant has been done. ► Deletion of the 35–42 peptide is the result of exon 6 skipping. ► A fast HPLC method for detecting αs1-CN genetic variants has been done. ► The short αs1-CN variant missing in Mediterranean occurs in milk of foreign breeds. A novel genetic variant at the αs1-casein locus of water buffalo (WB), 8-residue shorter than its wild-type has been found and sequenced. The internal deletion of the peptide E35KVNELsT42 was confirmed by the isolation of the junction peptide. The 8-residue deletion mutant has a molecular weight that is 919Da less than that of the wild-type. The novel isoform with a unique f35-42 deletion could be the result of the skipping of exon 6, generating an exon 6-deleted variant of αs1-casein. The wild-type and its shortened αs1-casein forms were found to co-exist in many individual milk samples. In contrast, the 8-residue, internally deleted αs1-casein variant did not occur in water buffaloes of the Mediterranean breed reared in Italy. Wild-type αs1-casein has 6 to 8 phosphate groups (P) while the internally deleted form 6 and 7P per molecule.