Top-Down proteomics based on LC-MS combined with cDNA sequencing to characterize multiple proteoforms of Amiata donkey milk proteins

•Donkey casein (CN) complexity, mainly due to intricate splicing events, has been deciphered.•Multiple αs2-CN with different C-terminal sequences, due to alternative splicing were found.•Definite evidence for the expression of a second gene encoding αs2-CN in the donkey genome.•New genetic variants for both CNs and whey proteins, have been identified.•Detection of a novel β-LG I isoform originating very likely from an original pattern of secretion.