Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry.
Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked beta-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.
Wang
Zihao
Z
Department of Biological Chemistry, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Udeshi
Namrata D
ND
O'Malley
Meaghan
M
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Hart
Gerald W
GW
D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation / Q000379:methods; D001921:Brain / Q000378:metabolism; D002846:Chromatography, Affinity; D006023:Glycoproteins / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods*; D051783:Methyl-CpG-Binding Protein 2 / Q000032:analysis / Q000378:metabolism; D015394:Molecular Structure; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D010777:Photochemistry / Q000379:methods; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D051381:Rats; D051843:Synucleins / Q000032:analysis / Q000378:metabolism; D038201:alpha-Crystallins / Q000032:analysis / Q000378:metabolism; D016875:tau Proteins / Q000032:analysis / Q000378:metabolism
D006023:Glycoproteins; C497394:Mecp2 protein, rat; D051783:Methyl-CpG-Binding Protein 2; D010455:Peptides; D011506:Proteins; D051843:Synucleins; D038201:alpha-Crystallins; D016875:tau Proteins; D000117:Acetylglucosamine
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