Enrichment of O-GlcNAc-modified peptides using novel thiol-alkyne and thiol-disulfide exchange

Hiroki Tsumoto; Daisuke Ogasawara; Noritaka Hashii; Takayoshi Suzuki; Yoshihiro Akimoto; Tamao Endo; Yuri Miura

doi:10.1016/j.bmcl.2015.04.082

Enrichment of O-GlcNAc-modified peptides using novel thiol-alkyne and thiol-disulfide exchange

Bioorg Med Chem Lett. 2015 Jul 1;25(13):2645-9. doi: 10.1016/j.bmcl.2015.04.082. Epub 2015 Apr 30.

Authors

Hiroki Tsumoto¹, Daisuke Ogasawara², Noritaka Hashii³, Takayoshi Suzuki², Yoshihiro Akimoto⁴, Tamao Endo¹, Yuri Miura⁵

Affiliations

¹ Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
² Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamohangi-cho, Sakyo-ku, Kyoto 606-0823, Japan.
³ Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
⁴ Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611, Japan.
⁵ 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.

PMID: 25980911
DOI: 10.1016/j.bmcl.2015.04.082

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.

Keywords: Enrichment; O-GlcNAc; Thiol-alkyne; Thiol-disulfide exchange.

MeSH terms

Acetylglucosamine / chemistry
Alkynes / chemistry
Amino Acid Sequence
Disulfides / chemistry*
Glycopeptides / chemistry*
Glycopeptides / isolation & purification
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Tandem Mass Spectrometry
alpha-Crystallins / chemistry

Substances

Alkynes
Disulfides
Glycopeptides
alpha-Crystallins
Acetylglucosamine