O-GlcNAcylation site mapping by (azide-alkyne) click chemistry and mass spectrometry following intensive fractionation of skeletal muscle cells proteins - Aix-Marseille Université Access content directly
Journal Articles Journal of Proteomics Year : 2018

O-GlcNAcylation site mapping by (azide-alkyne) click chemistry and mass spectrometry following intensive fractionation of skeletal muscle cells proteins

Abstract

The O-linked-N-acetyl-o-glucosaminylation (O-GlcNAcylation) modulates numerous aspects of cellular processes. Akin to phosphorylation, O-GlcNAcylation is highly dynamic, reversible, and responds rapidly to extracellular demand. Despite the absolute necessity to determine post-translational sites to fully understand the role of O-GlcNAcylation, it remains a high challenge for the major reason that unmodified proteins are in excess comparing to the O-GlcNAcylated ones. Based on a click chemistry approach, O-GlcNAcylated proteins were labelled with azid O-GalNAc and coupled to agarose beads. The proteome extracted from C2C12 myotubes was submitted to an intensive fractionation prior to azide-alkyne click chemistry. This combination of fractionation and click chemistry is a powerful methodology to map O-GlcNAc sites; indeed, 342 proteins were identified through the identification of 620 peptides containing one or more O-GlcNAc sites. We localized O-GlcNAc sites on proteins involved in signalling pathways or in protein modification, as well as structural proteins. Considering the recent role of O-GleNAcylation in the modulation of sarcomere morphometry and interaction between key structural protein, we focused on proteins involved in the cytoarchitecture of skeletal muscle cells. In particular, several O-GlcNAc sites were located into protein-protein interaction domains, suggesting that O-GlcNAcylation could be strongly involved in the organization and reorganization of sarcomere and myofibrils.
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hal-02143569 , version 1 (04-06-2019)

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Barbara Deracinois, Luc Camoin, Matthias Lambert, Jean -Baptiste Boyer, Erwan Dupont, et al.. O-GlcNAcylation site mapping by (azide-alkyne) click chemistry and mass spectrometry following intensive fractionation of skeletal muscle cells proteins. Journal of Proteomics, 2018, 186, pp.83-97. ⟨10.1016/j.jprot.2018.07.005⟩. ⟨hal-02143569⟩
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