Departments of †Chemistry, ‡Molecular and Cell Biology, and §Molecular Environmental Biology, University of California , Berkeley, California 94720, United States.
Howard Hughes Medical Institute and ⊥Department of Chemistry, Stanford University , Stanford, California 94305, United States.
J Am Chem Soc. 2016 Aug 31;138(34):10722-5. doi: 10.1021/jacs.6b03861. Epub 2016 Aug 17.
We report a non-destructive biochemical technique, termed "Glyco-seek", for analysis of O-GlcNAcylated proteins. Glyco-seek combines chemoenzymatic labeling, proximity ligation, and quantitative polymerase chain reaction to detect O-GlcNAcylated proteins with ultrahigh sensitivity. Our glycan-specific assay can be paired with traditional proximity ligation assays to simultaneously determine the change in total protein levels. We show that Glyco-seek detects attomoles of glycoproteins of interest from cell lysates, with sensitivity several orders of magnitude higher than that of current techniques. We used the method to directly assay the O-GlcNAcylation status of a low-abundance transcription factor from cell lysates without need for isolation or enrichment.
我们报告了一种称为“Glyco-seek”的非破坏性生化技术，用于分析 O-GlcNAc 修饰的蛋白质。Glyco-seek 结合了化学酶标记、邻近连接和定量聚合酶链反应，以超灵敏的方式检测 O-GlcNAc 修饰的蛋白质。我们的聚糖特异性测定法可以与传统的邻近连接测定法结合使用，以同时确定总蛋白质水平的变化。我们表明，Glyco-seek 可以从细胞裂解物中检测到纳摩尔数量的感兴趣的糖蛋白，其灵敏度比当前技术高几个数量级。我们使用该方法直接测定细胞裂解物中低丰度转录因子的 O-GlcNAc 化状态，而无需分离或富集。
