title: Purification and identification of O-GlcNAc-modified peptides using phosphate-based alkyne CLICK chemistry in combination with titanium dioxide chromatography and mass spectrometry.
abstract: A selective method for the enrichment of O-GlcNAcylated peptides using a novel CLICK chemistry reagent is described. Peptides modified by O-GlcNAc were enzymatically labeled with N-azidoacetylgalactosamine. The azide was then reacted with a phospho-alkyne using CLICK chemistry and O-GlcNAcGalNAzPO(4)-containing peptides were enriched using titanium dioxide chromatography. Modified peptides were analyzed using a combination of higher energy collision dissociation for identification and electron transfer dissociation to localize the site of O-GlcNAc attachment. The enrichment method was developed and optimized using an alpha-crystallin standard protein and then applied to a soluble protein preparation of mouse brain tissue and a nuclear preparation generated from HeLa cells. A total of 42 unique O-GlcNAcylated peptides were identified, including 7 novel O-GlcNAc sites.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Parker
authors.forename: Pankaj
authors.initials: P
authors.lastname: Gupta
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Cordwell
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Palmisano
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry*; D000480:Alkynes / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002845:Chromatography / Q000379:methods*; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D014025:Titanium / Q000737:chemistry*; D038201:alpha-Crystallins / Q000737:chemistry / Q000235:genetics
chemical_list: D000480:Alkynes; D009687:Nuclear Proteins; D010455:Peptides; D038201:alpha-Crystallins; C009495:titanium dioxide; D014025:Titanium; D000117:Acetylglucosamine