Kudelka Matthew R, Antonopoulos Aristotelis, Wang Yingchun, Duong Duc M, Song Xuezheng, Seyfried Nicholas T, Dell Anne, Haslam Stuart M, Cummings Richard D, Ju Tongzhong
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
Nat Methods. 2016 Jan;13(1):81-6. doi: 10.1038/nmeth.3675. Epub 2015 Nov 30.
Protein O-glycosylation has key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe an approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method used to amplify and profile mucin-type O-glycans synthesized by living cells. Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS). Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.
蛋白质O-糖基化在许多生物过程中发挥关键作用，但细胞合成的O-聚糖种类难以确定。在此，我们描述了一种称为细胞O-糖组报告/扩增（CORA）的方法，这是一种用于扩增和分析活细胞合成的粘蛋白型O-聚糖的灵敏方法。细胞将添加的全乙酰化苄基-α-N-乙酰半乳糖胺转化为多种修饰的O-聚糖衍生物，这些衍生物从细胞中分泌出来，便于通过高效液相色谱（HPLC）和质谱（MS）进行纯化分析。相对于传统的O-聚糖分析，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-聚糖种类。此外，当与计算模型相结合时，CORA可用于预测人类O-糖组的多样性，并为识别人类疾病的新型聚糖生物标志物提供新机会。
