An Isotope-Coded Photocleavable Probe for Quantitative Profiling of Protein O-GlcNAcylation

ACS Chem Biol. 2019 Jan 18;14(1):4-10. doi: 10.1021/acschembio.8b01052. Epub 2019 Jan 8.

Abstract

O-linked N-acetylglucosamine ( O-GlcNAc) is a ubiquitous post-translational modification of proteins and is essential for cell function. Quantifying the dynamics of O-GlcNAcylation in a proteome-wide level is critical for uncovering cellular mechanisms and functional roles of O-GlcNAcylation in cells. Here, we develop an isotope-coded photocleavable probe for profiling protein O-GlcNAcylation dynamics using quantitative mass spectrometry-based proteomics. This probe enables selective tagging and isotopic labeling of O-GlcNAcylated proteins in one step from complex cellular mixtures. We demonstrate the application of the probe to quantitatively profile O-GlcNAcylation sites in 293T cells upon chemical induction of O-GlcNAc levels. We further applied the probe to quantitatively analyze the stoichiometry of O-GlcNAcylation between sorafenib-sensitive and sorafenib-resistant liver cancer cells, which lays the foundation for mechanistic investigation of O-GlcNAcylation in regulating cancer chemoresistance. Thus, this probe provides a powerful tool to profile O-GlcNAcylation dynamics in cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / metabolism*
  • Cell Line, Tumor
  • HEK293 Cells
  • Humans
  • Isotope Labeling*
  • Molecular Probes / chemistry*
  • Protein Processing, Post-Translational
  • Proteins / metabolism*
  • Tandem Mass Spectrometry

Substances

  • Molecular Probes
  • Proteins
  • Acetylglucosamine