Cellular O-Glycome Reporter/Amplification to explore O-glycans of living cells.
Cellular O-Glycome Reporter/Amplification to explore O-glycans of living cells.
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DOI：
10.1038/nmeth.3675
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发表时间：
2016-01
期刊：
影响因子：
48
通讯作者：
Ju T
中科院分区：
文献类型：
--
作者：
Kudelka MR;Antonopoulos A;Wang Y;Duong DM;Song X;Seyfried NT;Dell A;Haslam SM;Cummings RD;Ju T
蛋白质o -糖基化在许多生物过程中起着关键作用，但细胞合成的o -聚糖库很难确定。在这里，我们描述了一种被称为细胞o -糖苷报告/扩增（CORA）的新方法，这是一种灵敏的方法，可以扩增和分析由活细胞合成的粘蛋白型o -糖苷。用过乙酰化苄基-α- n -乙酰半乳糖胺（GalNAc-α-苄基）培养的细胞将其转化为细胞分泌的多种修饰的o -聚糖衍生物，便于HPLC和质谱（MS）分析。与传统的o -聚糖分析相比，CORA的灵敏度提高了约100 - 1000倍，并在来自智人和小家鼠的十几种细胞类型中鉴定出更复杂的o -聚糖。此外，CORA与计算建模相结合，可以预测人类o型糖原的多样性，并为识别人类疾病的新型糖原生物标志物提供了新的机会。
Protein O-glycosylation plays key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe a new approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method to amplify and profile mucin-type O-glycans synthesized by living cells. Cells incubated with peracetylated benzyl-α-N-acetylgalactosamine (GalNAc-α-Benzyl) convert it to a large variety of modified O-glycan derivatives that are secreted from cells, allowing easy purification for analysis by HPLC and mass spectrometry (MS). CORA results in ~100–1000-fold increase in sensitivity over conventional O-glycan analyses and identifies a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, CORA coupled with computational modeling allows predictions on the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.