Detecting the "O-GlcNAc-ome"; detection, purification, and analysis of O-GlcNAc modified proteins.
The modification of Ser and Thr residues of cytoplasmic and nuclear proteins with a monosaccharide of O-linked beta-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.
Zachara
Natasha E
NE
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. nzachara@jhmi.edu
D000117:Acetylglucosamine / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D054794:Glycomics / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008954:Models, Biological; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
D006023:Glycoproteins; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D000117:Acetylglucosamine