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J Proteomics. 2018 Aug 30;186:83-97. doi: 10.1016/j.jprot.2018.07.005. Epub 2018 Jul 26.

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

Author information

1
Univ. Lille, EA 7369 - URePSSS - Unité de Recherche Pluridisciplinaire Sport Santé Société, F-59000 Lille, France.
2
Aix Marseille Univ, CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Marseille, France.
3
Univ. Lille, EA 7369 - URePSSS - Unité de Recherche Pluridisciplinaire Sport Santé Société, F-59000 Lille, France. Electronic address: caroline.cieniewski-bernard@univ-lille.fr.

Abstract

The O-linked-N-acetyl-d-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 azido-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-GlcNAcylation 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.

SIGNIFICANCE:

O-GlcNAcylation is an atypical glycosylation involved in the regulation of almost all if not all cellular processes, but its precise role remains sometimes obscure because of the ignorance of the O-GlcNAc site localization; thus, it remains indispensable to precisely map the O-GlcNAcylated sites to fully understand the role of O-GlcNAcylation on a given protein. For this purpose, we combined extensive fractionation of skeletal muscle cells proteome with click chemistry to map O-GlcNAc sites without an a priori consideration. A total of 620 peptides containing one or more O-GlcNAc sites were identified; interestingly, several of them belong to low expressed proteins, in particular proteins involved in signalling pathways. We also focused on structural proteins in view of recent data supporting the role of O-GlcNAcylation in the modulation of sarcomere cytoarchitecture; importantly, some of the O-GlcNAc sites were mapped into protein-protein interaction domains, reinforcing the involvement of O-GlcNAcylation in the organization and reorganization of sarcomere, and in larger extent, of myofibrils.

KEYWORDS:

Click chemistry; Fractionation; Mass spectrometry; O-GlcNAcylation; Post-translational modifications; Sites localization; Skeletal muscle cells

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