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

An in-depth molecular characterization of the main milk proteins, caseins (CNs) and whey proteins, from Amiata donkey combining top-down proteomic analysis (LC-MS) and cDNA sequencing, revealed multiple proteoforms arising from complex splicing patterns, including cryptic splice site usage and exon skipping events. Posttranslational modifications, in particular phosphorylation, increased the variety and complexity of proteoforms. αs2-CN perfectly exemplifies such a complexity. With 2 functional genes, CSN1S2 I and CSN1S2 II, made of 20 and 16 exons respectively, nearly 30 different molecules of this CN were detected in the milk of one Amiata donkey. A cryptic splice site usage, leading to a singular shift of the open reading frame and generating two αs2-CN I isoforms with different C-terminal sequences, was brought to light. Twenty different αs1-CN molecules with different phosphorylation levels ranging between 4 and 9P were identified in a single milk sample, most of them resulting from exon skipping events and cryptic splice site usage. Novel genetic polymorphisms were detected for CNs (β- and αs-CN) as well as for whey proteins (lysozyme C and β-LG I). The probable new β-LG I variant, with a significantly higher mass than known variants, appears to display an N-terminal extension possibly related to the signal peptide sequence. This represents the most comprehensive report to date detailing the complexity of donkey milk protein micro-heterogeneity, a prerequisite for discovering new elements to objectify the original properties of donkey’s milk.