Tsumoto Hiroki, Ogasawara Daisuke, Hashii Noritaka, Suzuki Takayoshi, Akimoto Yoshihiro, Endo Tamao, Miura Yuri
Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamohangi-cho, Sakyo-ku, Kyoto 606-0823, Japan.
Bioorg Med Chem Lett. 2015 Jul 1;25(13):2645-9. doi: 10.1016/j.bmcl.2015.04.082. Epub 2015 Apr 30.
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.
我们开发了一种用于富集O-连接的β-N-乙酰葡糖胺（O-GlcNAc）修饰肽段的选择性方法，该方法使用了新合成的硫醇-炔烃和硫醇-二硫键交换。首先，用含叠氮化物的GalNAc类似物对O-GlcNAc修饰肽段进行酶标记。然后，通过铜（I）催化的叠氮化物-炔烃环加成反应使叠氮基团与硫醇-炔烃反应。通过硫醇-二硫键交换，用硫醇反应性树脂富集硫醇修饰的肽段。从α-晶状体蛋白胰蛋白酶肽段中选择性分离出至少500飞摩尔的O-GlcNAc修饰肽段，并通过质谱检测。这种新颖的富集策略可用于生物样品的O-GlcNAc组分析。
