Optimization of 2-DE and multiplexed detection of O-GlcNAcome, phosphoproteome and whole proteome protocol of...

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.

This method permitted, after sequential image acquisition, the direct in-gel detection of O-GlcNAcome, phosphoproteome, and whole proteome of synapse-associated proteins.

This original method of differential proteomic analysis will permit to identify key markers of synaptic plasticity that are O-GlcNAcylated and/or phosphorylated, and their molecular regulations in neuronal processes.