TY - JOUR
T1 - Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry
AU - Wang, Zihao
AU - Udeshi, Namrata D.
AU - O'Malley, Meaghan
AU - Shabanowitz, Jeffrey
AU - Hunt, Donald F.
AU - Hart, Gerald Warren
PY - 2010/1
Y1 - 2010/1
N2 - Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked β-N-acetyl-glucosamine (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.
AB - Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked β-N-acetyl-glucosamine (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.
UR - http://www.scopus.com/inward/record.url?scp=76649126396&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=76649126396&partnerID=8YFLogxK
U2 - 10.1074/mcp.M900268-MCP200
DO - 10.1074/mcp.M900268-MCP200
M3 - Article
C2 - 19692427
AN - SCOPUS:76649126396
SN - 1535-9476
VL - 9
SP - 153
EP - 160
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
IS - 1
ER -