Top-Down proteomics based on LC-MS combined with cDNA sequencing to characterize multiple proteoforms of Amiata donkey milk proteins
| Autor: | Barbara Auzino, Guy Miranda, Céline Henry, Zuzana Krupova, Mina Martini, Federica Salari, Gianfranco Cosenza, Roberta Ciampolini, Patrice Martin |
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| Přispěvatelé: | Auzino, B., Miranda, G., Henry, C., Krupova, Z., Martini, M., Salari, F., Cosenza, G., Ciampolini, R., Martin, P. |
| Jazyk: | angličtina |
| Rok vydání: | 2022 |
| Předmět: |
Proteomics
DNA Complementary splicing variant Mass spectrometry Splicing variants Milk proteins Equidae Equus asinus High-performance liquid chromatography Whey Proteins Tandem Mass Spectrometry milk protein Equus asinus Milk proteins Splicing variants Exon skipping High-performance liquid chromatography Mass spectrometry casein phosphorylation Equus asinu Animals RNA Splice Sites casein phosphorylation mass spectrometry casein phosphorylation Exon skipping Chromatography Liquid Food Science |
| Popis: | 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 as2-CN I isoforms with different Cterminal sequences, was brought to light. Twenty different as1-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 (b- and as-CN) as well as for whey proteins (lysozyme C and b-LG I). The probable new -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. |
| Databáze: | OpenAIRE |
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