Mucin-type O-glycans decorate >80% of secretory and cell surface proteins and contribute to health and disease. However, dynamic alterations in the O-glycome are poorly understood because current O-glycomic methodologies are not sufficiently sensitive nor quantitative. Here we describe a novel isotope labeling approach termed Isotope-Cellular O-glycome Reporter Amplification (ICORA) to amplify and analyze the O-glycome from cells. In this approach, cells are incubated with Ac₃GalNAc-Bn (Ac₃GalNAc-[¹H₇]Bn) or a heavy labeled Ac₃GalNAc-Bn^D7 (Ac₃GalNAc-[²D₇]Bn) O-glycan precursor (7 Da mass difference), which enters cells and upon de-esterification is modified by Golgi enzymes to generate Bn-O-glycans secreted into the culture media. After recovery, heavy and light Bn-O-glycans from two separate conditions are mixed, analyzed by MS, and statistically interrogated for changes in O-glycan abundance using a semi-automated approach. ICORA enables ~100–1000-fold enhanced sensitivity and increased throughput compared to traditional O-glycomics. We validated ICORA with model cell lines and used it to define alterations in the O-glycome in colorectal cancer. ICORA is a useful tool to explore the dynamic regulation of the O-glycome in health and disease.

中文翻译：

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用细胞O-糖蛋白报道/扩增（ICORA）进行同位素标记以比较培养细胞的O-糖组学

粘蛋白型O聚糖修饰了80％以上的分泌蛋白和细胞表面蛋白，并促进了健康和疾病。但是，由于目前的O糖组学方法不够灵敏或定量，因此对O糖组的动态变化了解甚少。在这里，我们描述了一种新型的同位素标记方法，称为同位素细胞O型糖报道分子扩增（ICORA），可扩增和分析细胞中的O型糖。在这种方法中，将细胞与Ac ₃ GalNAc-Bn（Ac ₃ GalNAc- [ ¹ H ₇ ] Bn）或重标记的Ac ₃ GalNAc-Bn ^D7（Ac ₃ GalNAc- [ ² D ₇] Bn）O-聚糖前体（质量差异7 Da）进入细胞，并在去酯化后被高尔基酶修饰，生成分泌到培养基中的Bn-O-聚糖。回收后，将来自两个单独条件的重和轻Bn-O-聚糖混合，通过MS分析，并使用半自动方法对O-聚糖丰度的变化进行统计查询。与传统的O糖组学相比，ICORA可使灵敏度提高约100-1000倍，并提高通量。我们用模型细胞系验证了ICORA，并用它来定义大肠癌中O-糖基的变化。ICORA是探索健康和疾病中O糖组动态调节的有用工具。