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Detecting the “O-GlcNAcome”; Detection, Purification, and Analysis of O-GlcNAc Modified Proteins

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Glycomics

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 534))

Summary

The modification of Ser and Thr residues of cytoplasmic and nuclear proteins with a monosaccharide of O-linked β-N-acetylglucosamine is an essential and dynamic post-translational modification of metazoans. Deletion of the O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc, is lethal in mammalian cells highlighting the importance of this post-translational modification in regulating cellular function. O-GlcNAc is believed to modulate protein function in a manner analogous to protein phosphorylation. Notably, on some proteins O-GlcNAc and O-phosphate modify the same Ser/Thr residue, suggesting that a reciprocal relationship exists between these two post-translational modifications. In this chapter we describe the most robust techniques for the detection and purification of O-GlcNAc modified proteins, and discuss some more specialized techniques for site-mapping and detection of O-GlcNAc during mass spectrometry.

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Acknowledgements

The author would like to acknowledge Prof. Gerald W. Hart and Dr Chad Slawson (Johns Hopkins University School of Medicine) for comments on this manuscript; and the technical help of Katie Zoey Ho (Johns Hopkins Singapore). Some of the data presented in this paper were collected in the laboratory of Prof. Gerald W. Hart (Johns Hopkins University School of Medicine) and were supported by NIH grants HD R37-13563, CA R01-42486, DK-R01-61671, DK-R21/33-71280 (GWH), the National Heart, Lung, and Blood Institute, National Institutes of Health, contract No. N01-HV-28180 (GWH). Under a licensing agreement between Covance Research Products and The Johns Hopkins University, Dr. Hart receives a share of royalties received by the university on sales of the CTD 110.6 antibody. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with its conflict of interest policies. NEZ is supported by an A*Star Research grant to Johns Hopkins Singapore.

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Authors and Affiliations

  1. Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Natasha E. Zachara

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  1. Natasha E. Zachara
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Correspondence to Natasha E. Zachara .

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Editors and Affiliations

  1. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

    Nicolle H. Packer

  2. Centre for Bioanalytical Science, Chemistry Department, NUI Galway, Galway, Ireland

    Niclas G. Karlsson

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Zachara, N.E. (2009). Detecting the “O-GlcNAcome”; Detection, Purification, and Analysis of O-GlcNAc Modified Proteins. In: Packer, N.H., Karlsson, N.G. (eds) Glycomics. Methods in Molecular Biology™, vol 534. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-022-5_19

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  • DOI: https://doi.org/10.1007/978-1-59745-022-5_19

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-774-7

  • Online ISBN: 978-1-59745-022-5

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