Identifying potentially O-GlcNAcylated proteins using metabolic labeling, bioorthogonal enrichment, and Western blotting

Methods Enzymol. 2019:622:293-307. doi: 10.1016/bs.mie.2019.02.017. Epub 2019 Mar 6.

Abstract

O-GlcNAcylation is a widespread posttranslational modification of intracellular proteins. Phenotypic and genetic experiments have established key roles for O-GlcNAc in development, mammalian cell survival, and several human diseases. However, the underlying mechanisms by which this modification alters biological pathways are still being discovered. An important part of this discovery process is the discovery of O-GlcNAcylated proteins, where chemical approaches have been particularly powerful. Here we describe how to combine one of these approaches, metabolic chemical reporters (MCRs), with bioorthogonal chemistry and Western blotting to identify potentially O-GlcNAcylated proteins.

Keywords: Bioorthogonal; Biotin enrichment; Click chemistry; Metabolic chemical reporter; O-GlcNAc.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acetylglucosamine / analysis*
  • Acetylglucosamine / metabolism
  • Animals
  • Blotting, Western / methods*
  • Click Chemistry / methods
  • Electrophoresis, Polyacrylamide Gel / methods
  • Glycosylation
  • Humans
  • Protein Processing, Post-Translational
  • Proteins / chemistry*
  • Proteins / metabolism

Substances

  • Proteins
  • Acetylglucosamine