title: High-sensitivity FAB-MS strategies for O-GlcNAc characterization.
abstract: In this paper we report the first application of fast atom bombardment mass spectrometry (FAB-MS) to O-linked N-acetylglucosamine (O-GlcNAc)-bearing glycopeptides. Using N-acetylgalactosamine (GalNAc)- and Gal-GalNAc-containing glycopeptides (isolated from Tn glycophorin and desialylated normal glycophorin, respectively) as readily available model compounds, rapid and sensitive derivatization/FAB-MS strategies applicable to serine/threonine-rich glycopeptides have been devised. Peptides and glycopeptides were propionylated in a 1 min reaction using a mixture of trifluoroacetic anhydride and propionic acid, and the product mixtures were analysed directly by FAB-MS. Glycopeptides and peptides rich in hydroxylated residues afforded characteristic clusters of molecular ions at high sensitivity. Additional sensitivity enhancement was achieved by prior esterification of carboxyl groups. These methods were used in a study of O-GlcNAc glycopeptides produced by purified O-GlcNAc transferase addition of GlcNAc to the synthetic peptides YSDSPSTST and YSGSPSTST in which Y is tyrosine, S is serine, D is aspartic acid, P is proline, T is threonine and G is glycine. The propionyl derivatives afforded high-quality spectra which unequivocally showed that the majority of the glycopeptides were substituted with a single GlcNAc residue. Low pmol quantities of material gave detectable signals. The propionylation/FAB-MS procedure has been combined with gas-phase sequencing strategies and shows promise for defining the sites of glycosylation of O-GlcNAc glycopeptides that are available in limited quantities.
authors.affiliation: Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: Reason
authors.forename: I P
authors.initials: IP
authors.lastname: Blench
authors.forename: R S
authors.initials: RS
authors.lastname: Haltiwanger
authors.forename: G W
authors.initials: GW
authors.lastname: Hart
authors.forename: H R
authors.initials: HR
authors.lastname: Morris
authors.forename: M
authors.initials: M
authors.lastname: Panico
authors.forename: A
authors.initials: A
authors.lastname: Dell
mesh_terms: D000116:Acetylgalactosamine / Q000032:analysis*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002240:Carbohydrate Sequence; D006020:Glycopeptides / Q000737:chemistry* / Q000378:metabolism; D006021:Glycophorins / Q000737:chemistry*; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009439:Neuraminidase; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D012694:Serine; D016339:Spectrometry, Mass, Fast Atom Bombardment / Q000379:methods; D013912:Threonine
chemical_list: D006020:Glycopeptides; D006021:Glycophorins; D007202:Indicators and Reagents; D009842:Oligopeptides; D013912:Threonine; D012694:Serine; D009439:Neuraminidase; D000116:Acetylgalactosamine