Nucleocytoplasmic glycosylation, O-GlcNAc: identification and site mapping.
beta-O-linked N-acetylglucosamine (O-GlcNAc) is posttranslationally added to serine and threonine residues of many nuclear and cytoplasmic proteins found in metazoans. This modification is dynamic and responsive to numerous stimuli and conditions, suggesting an important role in many regulatory pathways. Moreover, the O-GlcNAc modification seems to compete with phosphorylation for sites of attachment, indicating a reciprocal relationship with phosphorylation. This chapter includes protocols for: (1) identifying the O-GlcNAc modification on proteins through immunoblotting, lectin affinity chromatography, and galactosyltransferase labeling; and (2) identifying and enriching for the sites of attachment using the mass spectrometry-based beta-elimination followed by Michael addition with dithiothreitol (BEMAD) technique.
Zachara
Natasha Elizabeth
NE
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Cheung
Win Den
WD
Hart
Gerald Warren
GW
D000117:Acetylglucosamine / Q000032:analysis* / Q000378:metabolism; D000906:Antibodies / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism*; D005700:Galactosyltransferases / Q000737:chemistry; D006031:Glycosylation; D015151:Immunoblotting / Q000379:methods; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D014909:Wheat Germ Agglutinins / Q000737:chemistry; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry
D000906:Antibodies; D009687:Nuclear Proteins; D011506:Proteins; D014909:Wheat Germ Agglutinins; D005700:Galactosyltransferases; D001619:beta-N-Acetylhexosaminidases; D000117:Acetylglucosamine