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Mass Spectrometry-Based Quantitative O-GlcNAcomic Analysis

  • Junfeng Ma
  • Gerald W. HartEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1410)

Abstract

The dynamic co- and post-translational modification (PTM) of proteins, O-linked β-d-N-acetylglucosamine modification (O-GlcNAcylation) of serine/threonine residues is critical in many cellular processes, contributing to multiple physiological and pathological events. The term “O-GlcNAcome” refers to not only the complete set of proteins that undergo O-GlcNAcylation but also the O-GlcNAc status at individual residues, as well as the dynamics of O-GlcNAcylation in response to various stimuli. O-GlcNAcomic analyses have been a challenge for many years. In this chapter, we describe a recently developed approach for the identification and quantification of O-GlcNAc proteins/peptides from complex samples.

Key words

Chemoenzymatic labeling Electron transfer dissociation (ETD) GalT1 labeling O-GlcNAcylation O-GlcNAcome Photocleavage Site mapping SILAC Quantitative mass spectrometry 

Notes

Acknowledgments

Original research in this work was supported by NIH P01HL107153, R01DK61671, and NIH N01-HV-00240 (to G.W.H.). We appreciate Dr. Zihao Wang for his significant contribution for the initial development of this protocol and for his critical reading of the manuscript. Helpful discussion from the Hart laboratory is acknowledged. We also thank Drs. Feng Yang and Richard Smith and their coworkers at the Pacific Northwest National Laboratory (Richland, WA) for valuable comments of the enrichment protocol.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biological ChemistryThe Johns Hopkins University School of MedicineBaltimoreUSA

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