Optimization of 2-DE and multiplexed detection of O-GlcNAcome, phosphoproteome and whole proteome protocol of synapse-associated proteins within the rat sensorimotor cortex

Julie Fourneau (1) , Caroline Cieniewski-Bernard (1) , Marie-Hélène Canu (1) , Sophie Duban-Deweer (2) , Johann Hachani (2) , Bruno Bastide (1) , Erwan Dupont (1)

1 URePSSS - Unité de Recherche Pluridisciplinaire Sport, Santé, Société (URePSSS) - ULR 7369 - ULR 4488
2 LBHE - Laboratoire de la Barrière Hémato-Encéphalique

Several studies have shown the importance of phosphorylation, O-GlcNAcylation and their interplay in neuronal processes.To get understanding about molecular mechanisms of synaptic plasticity, we performed a preparation of synaptic protein-enriched fraction on a small sample of rat sensorimotor cortex. We then optimized a multiplexed proteomic strategy to detect O-GlcNAcylated proteins, phosphoproteins, and the whole proteome within the same bidimensional gel. We compared different protocols (solubilisation buffer, reticulation and composition of the gel, migration buffer) to optimize separating conditions for 2D-gel electrophoresis of synaptic proteins. The O-GlcNAcome was revealed using Click chemistry and the azide-alkyne cycloaddition of a fluorophore on O-GlcNAc moieties. The phosphoproteome was detected by Phospho-Tag staining, while the whole proteome was visualized through SYPRORuby staining.

Keywords: 2-DE; O-GlcNAcylation; Phosphorylation; Proteomic; Synapse-associated proteins

Domains: Life Sciences [q-bio]

Julie Fourneau, Caroline Cieniewski-Bernard, Marie-Hélène Canu, Sophie Duban-Deweer, Johann Hachani, et al.. Optimization of 2-DE and multiplexed detection of O-GlcNAcome, phosphoproteome and whole proteome protocol of synapse-associated proteins within the rat sensorimotor cortex. Journal of Neuroscience Methods, 2020, 343, pp.108807. ⟨10.1016/j.jneumeth.2020.108807⟩. ⟨hal-03127213⟩