Top-Down proteomics based on LC-MS combined with cDNA sequencing to characterize multiple proteoforms of Amiata donkey milk proteins
Auzino, Barbara | Miranda, Guy | Henry, Céline | Krupova, Zuzana | Martini, Mina | Salari, Federica | Cosenza, Gianfranco | Ciampolini, Roberta | Martin, Patrice
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. Post-translational 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.
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