title: Enrichment of O-GlcNAc-modified peptides using novel thiol-alkyne and thiol-disulfide exchange.
abstract: We have developed a selective method for the enrichment of O-linked β-N-acetylglucosamine (O-GlcNAc)-modified peptides, which uses a newly synthesized thiol-alkyne and a thiol-disulfide exchange. First, O-GlcNAc-modified peptides were enzymatically labeled with an azide-containing GalNAc analog. Then, the azide moiety was reacted with thiol-alkyne through a copper(I)-catalyzed azide-alkyne cycloaddition. The thiol-modified peptides were enriched with thiol-reactive resin through a thiol-disulfide exchange. At least 500fmol of O-GlcNAc-modified peptides was selectively isolated from α-crystallin tryptic peptides and detected by mass spectrometry. This novel enrichment strategy could be used for O-GlcNAcome analysis of biological samples.
authors.affiliation: Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tsumoto
authors.affiliation: Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamohangi-cho, Sakyo-ku, Kyoto 606-0823, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ogasawara
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Noritaka
authors.initials: N
authors.lastname: Hashii
authors.affiliation: Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamohangi-cho, Sakyo-ku, Kyoto 606-0823, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Akimoto
authors.affiliation: Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
authors.forename: Tamao
authors.initials: T
authors.lastname: Endo
authors.affiliation: Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan. Electronic address: miura@center.tmig.or.jp.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Miura
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry; D000480:Alkynes / Q000737:chemistry; D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D038201:alpha-Crystallins / Q000737:chemistry
chemical_list: D000480:Alkynes; D004220:Disulfides; D006020:Glycopeptides; D038201:alpha-Crystallins; D000117:Acetylglucosamine
keywords: Enrichment; O-GlcNAc; Thiol-alkyne; Thiol-disulfide exchange